Francisco Pacheco (1564–1644) was one of the most important Spanish painters, the author of the famous treatise Art of Painting, and a founder of Seville art school. His painting La Inmaculada (1610) forms part of the important art collection in the Archbishop’s Palace in Seville and represents the first realization of a new iconography, established by Pacheco. Later, he carried out several paintings with the same subject which are today in different collections. As part of a larger project, La Inmaculada was recently restored and analysed. The characterization of materials and painting procedures was carried out applying noninvasive in situ analysis by ultraviolet fluorescence (UVF), infrared reflectography (IRR), and X-ray fluorescence spectrometry (XRF). Results revealed many retouches, probably from different periods, as well as some pentimenti in the composition. Chemical analysis indicates a preparation made of Seville clay, lead white and some calcite on an animal glue layer, according to Pacheco’s own treatise, and a corresponding pigment palette for the 17th century: lead white, yellow and red earths, lead–tin yellow, vermilion, azurite, smalt, a copper-based green, umber, and bone black. Furthermore, modern pigments such as titanium and zinc whites, chrome green, and cadmium yellow were also identified as results of later interventions.