Two chromatographic methods have been compared for analysis of protein-binding media used in paintings, namely, HPLC with fluorescence detection and GC-MS. The proteins were hydrolyzed to the corresponding amino acids (AAs) by gaseous HCl and the AAs were derivatized with methyl chloroformate, followed by GC-MS or by HPLC after derivatization with the AccQ fluorescence reagent. The hydrolysis, derivatization reactions and the chromatographic procedures have been optimized and applied to standard binding media, model and real samples of paintings. The methods have been compared and critically evaluated.