Art and liturgical practices in the monastery of Descalzas Reales in Madrid, second half of the 16th century and 17th century, addresses the analysis of the different ceremonies carried out by the religious community in relation to their liturgical calendar. The significant collection of images, liturgical objects and sacred vestiges that this foundation of Colettines came to treasure gave meaning to the different spaces inhabited by the nuns, and to the variety of daily liturgical practices. Since the founder of this royal patronage, Joanna of Austria (1535-1573), entrusted most of her assets to the monastery, various Habsburg women, such as her sister, the Empress Mary, and her niece, Sister Margarita de la Cruz, contributed to extending and altering the legacy of the Princess of Portugal. This collection of objects and images was used by a community of Poor Clares arriving from the Colettine diaspora, whose seed we find in the Monastery of Santa Clara de Gandía. In turn, these nuns will show a strong commitment to their origins, as well as to the devotional practices and customs established by the first inhabitants of the Descalzas Reales according to the rituality practiced at the motherhouse. Both factors converge in this Madrid monastic space, so that the different liturgical practices conducted there and the processes of veneration and accommodation of images and relics acquire different meanings that we try to analyse in the course of our study.The archive of the Monastery of Las Descalzas Reales in Madrid currently preserves an interesting collection of documents from different types of sources that allow us to approach its history from different points of view and through different realities. The comparative analysis of a selection of sources such as their Constituciones or rules of foundation, together with the manuscripts produced by the nuns themselves with regard to their daily life and other sources such as inventories, allows us to complement and delimit our look into the cloister. Even though their origins and purpose are different, these three documentary typologies are part of the same project that directly concerns the subject of our study: the creation of a ceremonial framework for cloistered life, as well as its development through the visual collection and liturgical practices of the monastery. Liturgical practices gave meaning and significance to the combination of all the images, relics, objects, readings, and spaces used by the religious community in its daily life.