The first art collections of the Medici family, the result of their patronage towards the artists of their day, dates from the early 15th century, with Cosimo the Elder, Piero the Gouty and Lorenzo the Magnificent. The sacking of Palazzo Medici in 1494, which took place after Piero the Unfortunate had been thrown out of the city, was responsible for the loss of most of these works of art but, when the cadet branch of the family came to power in 1537, Cosimo I, son of Giovanni dalle Bande Nere, set to work to retrieve as many of the treasures still in the city as possible. For example, the vases in pietra dura that once belonged to Lorenzo the Magnificent, now in the Silver Museum at the Pitti Palace, are the result of this. Cosimo I also managed to trace Paolo Giovio's collection of portraits of famous men, which are of great historical interest (now exhibited along the corridors of the Uffizi Gallery). Two works by Titian are also included in his inventories: the portraits of Pietro Aretino and Cardinal Ippolito de' Medici.

Although it is thanks to Francesco I that the Gallery was created inside the Palace of the Uffizi, it was actually his brother Ferdinando I who enriched it with many splendid antique sculptures and important paintings like the Madonna of the Chair by Raphael (exhibited in the Tribune from 1589 and which he probably bought a few years earlier when he was a Cardinal in Rome). The Grand Duke also did his best to make sure that the city's artistic heritage was not scattered and on October 24th 1602 drew up a law to limit the exportation of paintings: "so that they do not go away and leave the City without their ornament".

The first guide to the city, "The beauties of Florence", containing a description of all the works of art in the private collections in Florence, was published in 1591 by Bocchi, during the reign of Ferdinando I (1587-1609). His son Cosimo II (1590-1621) specialized in collecting paintings on stone and rare materials (like slate) as well as forming a collection of small paintings by Italian and foreign artists: Filippo Napoletano, Callot, Ligozzi, Poelenburgh. However he preferred to exhibit these in a small loggia at the Pitti Palace.

There were some exceptions, like the Madonna and Child by Correggio, which was given to him by the Duke of Modena in 1617, and the Veiled lady by Raphael, which arrived at the court of Cosimo II in 1620 as part of a legacy left by the Botti family. The following year, when Ferdinando II came to the throne, was to mark the beginning of the golden century of the Medici art collections (1621-1737).