The coupling of molecular analyses with the conservation of artworks has gained growing importance over the last decades, and today it can take advantage of state-of-the-art analytical techniques, such as mass spectrometry-based proteomics. This technique allows for the deep characterisation of proteins with high accuracy and reliability. Protein-based binders are among the most common organic artistic materials, and have been used in art production for centuries. However, the applications of proteomic techniques to these materials are still limited. In this work, a palaeoproteomic workflow was successfully tested on a set of paint reconstructions, and subsequently applied to micro-samples from a 15th-century panel painting, attributed to Sandro Botticelli’s workshop. The method allowed for confident identification of the protein-based binders and their biological origin in all historical micro-samples, as well as for the discrimination of the binder used in the ground and paint layers. These results were discussed thoroughly in order to outline a set of guidelines, covering the main steps of the data analysis of protein sequencing results from an artwork. This work is the first application of the adopted palaeoproteomic protocol to pigmented materials, with results showing that the approach is accurate, highly sensitive, and broadly applicable in the cultural heritage field, due to the limited amount of starting material it requires.