The characterization of the organic components in a complex, multilayered paint structure is fundamental for studying painting techniques and for authentication and restoration purposes. Proteinaceous materials, such as animal glue, are of particular importance since they are widely used as binders, adhesives and for gilding. Even though proteins are usually detected by chromatographic and proteomic techniques, immunological methods represent an alternative powerful approach to protein analysis thanks to the high specificity of antigen–antibody reactions. Our previous studies demonstrated that ovalbumin and casein could be localized in paint cross-sections with high sensitivity and good spatial resolution (i.e. within the single painting layers) by using chemiluminescent (CL) immunochemical microscope imaging. In the present research work, we describe for the first time the immunolocalization of collagen (the main protein of animal glue) in paint cross-sections by CL imaging microscopy. Two different analytical protocols have been developed, allowing either the detection of collagen or the simultaneous detection of collagen and ovalbumin in the same paint sample. The assays were used to detect collagen and ovalbumin in cross-sections from model samples and historical paintings (a wall painting dated to 1773–1774 and a painted wood panel of the Renaissance period) in order to achieve information on paint techniques and past restoration interventions. Figure Left Reflected light image of a cross-section of a sample taken from a Renaissance painted wood panel. Right Localization of the proteins collagen (from animal glue) and ovalbumin in a painting cross-section assessed by multiplexed chemiluminescence immunochemical imaging (the chemiluminescent signals corresponding to collagen and ovalbumin are displayed in shades of blue and red , respectively)