Chemistry is at the front line of preservation of work of arts. Reliable but simple protocols for localizing proteins in art samples are necessary to guide conservators in their work. In this article, we investigated the interaction between some commonly used fluorescent stains and protein-based paint binders. Beside fluorescence, we used for the first time circularly polarized luminescence (CPL) as a tool to reveal interactions in complex matrices relevant for the field of cultural heritage. We show that, in such cases, CPL provides unique information compared to more common techniques such as fluorescence and circular dichroism. These findings will contribute to provide a rational base to empirical observations in staining procedures, even in arduous and complex cases.