By about 1854, part-owner Capt. J. D. Webster, later General Webster and chief of staff at the Battle of Shiloh, and Dr. Charles H. Ray of Galena, Illinois through Horace Greeley convinced Joseph Medill of Cleveland's Leader to become managing editor. Ray became editor-in-chief, Medill became the managing editor, and Alfred Cowles, Sr., brother of Edwin Cowles, initially was the bookkeeper. Each purchased one third of the Tribune.Under their leadership the Tribune distanced itself from the Know Nothings and became the main Chicago organ of the Republican Party.However, the paper continued to print anti-Catholic and anti-Irish editorials.The Tribune absorbed three other Chicago publications under the new editors: the Free West in 1855, the Democratic Press in 1858, and the Chicago Democrat in 1861, whose editor, John Wentworth, left his position to become Chicago Mayor. Between 1858 and 1860, the paper was known as the Chicago Press & Tribune. After November 1860 it became the Chicago Daily Tribune.Before and during the American Civil War, the new editors pushed an abolitionist agenda and strongly supported Abraham Lincoln, whom Medill helped secure the Presidency in 1860. The paper remained a force in Republican politics for years afterwards.

In 1861 the Tribune published new lyrics for the song "John Brown's Body" by William W. Patton, rivaling the ones published two months later by Julia Ward Howe. Medill served as mayor of Chicago for one term after the Great Chicago Fire of 1871.

Under the 20th Century editorship of Colonel Robert R. McCormick the paper was strongly isolationist and actively biased in its coverage of political news and social trends, calling itself, "The American Paper for Americans," excoriating the Democrats and the New Deal, resolutely disdainful of the British and French and greatly enthusiastic for Chiang Kai-shek and Sen. Joseph McCarthy.

When McCormick took over as co-editor (with his cousin Joseph Patterson) in 1910, the Tribune was the 3rd best selling paper among Chicago's eight dailies, with a circulation of only 188,000.[15] The young cousins added features such as advice columns and homegrown comic strips like Little Orphan Annie and Moon Mullins, then turned to "crusades", with their first success coming with the ouster of the Republican political boss of Illinois, Senator William Loring.[15] At the same time, the Tribune competed with the Hearst paper, the Chicago Examiner, in a circulation war. By 1914, the cousins succeeded in forcing out Managing Editor William Keeley. By 1918, the Examiner was forced to merge with the Chicago Herald.

In the 1920s, Patterson left to take over the editorship of his own paper, the New York Daily News.In a renewed circulation war with Hearst's Herald-Examiner, McCormick and Hearst ran rival lotteries in 1922. The Tribune won the battle, adding 250,000 readers to its ranks. During the McCormick years, the Tribune was a champion of modified spelling (such as spelling "although" as "altho").

McCormick, a vigorous campaigner for the Republican Party, died in 1955, just four days before Democratic boss Richard J. Daley was elected mayor for the first time.

One of the great scoops in Tribune history came when it obtained the text of the Treaty of Versailles in June 1919. Another was its revelation of United States war plans on the eve of the Pearl Harbor attack. Its June 7, 1942 front page announced that America had broken Japan's naval code.