The article describes the history of higher education in Florence from the beginning of the twentieth century until the mid-1920s, when the ancient Institute of Higher Studies, founded in 1859, was transformed into a modern University. The development of the university institution is reconstructed through the detailed analysis of the annual reports presented by the superintendents of the Institute and making use of the unpublished documents preserved in the historical archives of the University. Particular attention is paid to the evolution of the number of enrolled students and graduates, as well as to the expansion of the training offer made possible by the opening of new faculties and new laboratories. Finally, the article focuses on some important professors, such as the physicist Antonio Garbasso who was also mayor of Florence, and the historian Gaetano Salvemini who was one of the leaders of the anti-fascist movement and, due to his political positions, in 1925 he was forced to leave the University to emigrate to the United States.