The study of proteins in cultural heritage is an analytical challenge for many reasons: samples are available in tiny amounts, and the proteins are encased in complex solid media and modified by ageing and other external conditions. This paper presents the development of new methodologies based on proteomic techniques underlining the critical steps: the protein extraction, the technique adaptations to very low sample quantities and the protein identifications despite their ageing and denaturation. These techniques were applied to art and archaeological samples, allowing for the first time the identification of proteins in binding media in Renaissance art paintings and in potsherds from coastal archaeological sites.