Profiles in Courage articulates and argues for the significance of the idea of "political courage" in American political history. Through four parts, the author, President John F. Kennedy argues that the preeminent value of a senator is "political courage," which he defines from drawing from the lives of eight former American senators.

The first of these Senators is John Quincy Adams, the son of President John Adams. Considering an embargo against Britain over its aggression on American shipping, John Quincy Adams falls out of favor with his own Federalist Party. While an embargo would place great strain on his constituents' businesses, Adams believes that national defense trumps these concerns. Supporting the embargo, he loses much of his support, and is branded a "traitor." However, John Quincy Adams' independent and exacting character helps him return to the House of Representatives; later, he becomes the US’s ninth president. 

Kennedy next investigates the Compromise of 1850, and the intensifying conflict between North and South. Kennedy focuses on three Senators: Daniel Webster of Massachusetts, Thomas Benton of Missouri, and Samuel Houston of Texas. Kennedy details and applauds these senators—abolitionists and slaveowners alike—in their attempt to preserve the Union by reaching compromise. For each of these senators, Kennedy makes clear the immense sacrifice in their political careers and ambitions and challenging their own deeply-held convictions in the name of national interest. This highlights a claim made by Kennedy in the Introduction, which argues that compromise is an unavoidable part of politics—particularly American politics—while at the same time providing the caveat that the value and rightness of compromise cannot be easily or safely predicted. In addition to this, Part 2 serves to illustrate what Kennedy believes is the toxic effect of regionalism and partisanship on American politics, and with it, the added necessity of political courage. The nearing possibility of civil war causes senators not only to break with their friends and colleagues, but also to challenge their most deeply-held convictions, in order to preserve the Union. 

In Part 3, Kennedy takes up the post-Civil War period of American politics. He remarks on how the expansion of the country brought with it both the trivialization of Senate business and the rise of partisan politics, in a regionally-defined political landscape. Kennedy's first example of the overreach of partisan politics is the 1868 impeachment of Andrew Johnson. 

The immediate political issue at stake was the Reconstruction of the South, following its defeat in the Civil War. Andrew Johnson, a Democrat, was accused of attempting to weaken and remove important provisions of civil rights, provisions that the Republican Party wanted. Amidst this, Johnson was accused of improperly removing his secretary of the US Navy and the Republican Party organizes an attempt to remove the Johnson from office using Articles of Impeachment. 

However, the trial process itself exposes the deeply partisan motivations of the Republican Party in pushing for President Johnson's removal from office; the president opposed many of the policies supported by the Republican Party, some of them deemed harsh against Southern states. One Republican senator, Edmund Ross of Kansas, is so disturbed by these attempted actions he refuses to convict Johnson, whose presidency is saved by one vote. Ross, a fervent abolitionist and veteran of the Union Army, is branded a traitor to his ideals; his political career never recovers, though he does not regret the decision. 

Roughly ten years later, Senator Lucius Lamar, a Democrat from Mississippi, is faced with a similar dilemma in matters of monetary policy. When the Democratic Party en masse joins the cause of "free silver," a measure intended to ease monetary policy and help indebted Southern farmers by buying silver and coining it, Senator Lamar breaks with his party, owing to what he believes are critical deficiencies and problems of the "free silver" movement. Like Ross, he is scorned by his party leaders. After undertaking a grueling and ambitious tour of his home state, Mississippi, he likens his senatorial duty to a sailor in a "topmost sail" (160), sent to see what others cannot. 

In Part 4, Kennedy focuses on America's new role on the international stage, and the troubling implications thereof. The Senate has changed; thanks to the Seventeenth Amendment, America's senators are now directly elected by their states. Alongside this spirit of populism arrive new dangers and anxieties. The most dangerous of these is America's entry into World War I. In 1914, Nebraska Senator George Norris' attempts to filibuster a measure to arm American shipping. George Norris, a pacifist, believes this measure to be a way for President Woodrow Wilson to skirt around Congress's capacity to declare war. Although Norris successfully filibusters the measure to arm American ships, Wilson ultimately circumvents Congress. The reaction is swift, and harsh: Senator Norris is branded a traitor, and condemned by even his own Republican party. Like Senator Lamar a half-century prior, he returns to his home state and successfully defends himself to his constituents, earning their trust. Norris faces other challenges and hard choices in the Senate, meeting them with success and failure. However, he distinguishes himself through his integrity.

The last senator described in detail in Profiles in Courage is Robert A. Taft, the son of twenty-seventh President William Howard Taft. Following the end of World War II, Taft speaks out against the institutionalization of ex post facto punishment of Nazi war criminals. His reasoning is that to hold individuals accountable for heretofore-undefined crimes is a dangerous expansion of court powers, one that contravenes the Constitution. His statements cause an immediate uproar; Senator Taft is accused of being a Nazi sympathizer, and is denounced even by members of his own party. According to Kennedy, however, Taft does illustrate the ambiguity of the system of international law, one which the scale and scope of the Nazis' atrocities trivialized. For Kennedy, this ideal of courage is less linked with actual policy and more closely aligned with exercising one’s individual's conscience in the face of political and social pressure. Kennedy believes the changing role of the Senate, from a debating and advisory chamber for select statesmen to a legislature body in its own reflect, nevertheless requires such acts of conscience. Kennedy also believes the exercise and protection of conscience is not primarily the obligation of politicians, but a duty shared by all citizens of a free society.