The entire southern third of the structure is taken up by the monastery. Although the cloistered Augustinian Fathers have lived there since 1885, Hieronymite monks first inhabited there in 1567. One of the outstanding representations of the architecture of the Monastery, the Patio de los Evangelistas, a masterpiece created by Juan Bautista de Toledo, serves as the focal point of the enclosure. The magnificent main staircase that connects its two floors has frescoes by Luca Giordano on its vaults. Luca Cambiaso and Pellegrino Tibaldi carried on Luca Cambiaso's ambitious graphic concept for its colonnades. Juan de Herrera, who was inspired by Bramante's Tempietto of San Pietro in Montorio, created a stunning temple in the center of the cloister out of granite, marble, and jasper in a variety of colors. Juan Bautista Monegro chiseled the sculptures of the four Evangelists from a single block of marble, and they each hold an open book with a passage from their Gospel printed in the language in which it was originally written.

The Lower Priory Cell, which is distinct from the Chapter Rooms next to it, features a painting on the ceiling of The Judgement of Solomon by Francesco da Urbino, which serves as a reminder to the prior of the necessity of having just administration at the helm of the Monastery. The Adoration of the Sacred Form by Claudio Coello can be found in the sacristy, which is still in use. The Martyrdom of Saint Lawrence by Titian, one of the great works of the Italian Renaissance, is kept in the Old or Borrowed Church. Philip II had it painted for the main altarpiece of the Basilica but decided against it because of its dark coloring, which made it difficult to see from a distance.