Two of the most serious conditions affecting the plaster and murals in the dome of the chapel are delamination and detachment, which can lead to major areas of loss. Water ingress from above has clearly been a problem for many years, but in order to understand the intrinsic factors related to this detachment it was necessary to conduct lab analysis to understand the physical and chemical composition of the substrate, binders, and pigments used in the various mural campaigns. Both the enfoscado or scratch coat and the enlucido or finish coat of plaster were physically analyzed using acid digestion, sieve analysis, microscopic examination, and XRD (X-ray diffraction). The enfoscado was found to have a very low binder to non-binder ratio, lending it insufficient cohesive strength, and likely contributing to failure. In order to analyze the stratigraphy of the Rosario mural campaigns, cross sections of selected samples were examined in order to determine the number and sequence of paint layers and other characteristics. The binders and pigments of the paints themselves were also analyzed using Energy Dispersive Spectroscopy (EDS), which identifies the elemental composition of a sample, and Fourier Transform Infrared Spectroscopy (FTIR), which was used to identify organic binding material. Pigment composition offered important clues to the dating of the various campaigns.