Polyphenols can be widely found in plants and plant-based food. Polyphenols with an ortho-dihydroxy structure, such as caffeic acid (CA), can easily be oxidized to their ortho-quinones. These quinones can react with nucleophilic side chains of proteins, such as the thiol group in cysteine (Cys), and form adducts that can affect the structure and function of these proteins. Individual adducts have been described qualitatively, but quantitative analysis has been hampered by the poor stability of the adducts during sample preparation. In this study, 2-S-cysteinyl caffeic acid was synthesized either in a free or protein-bound form. A one-enzyme hydrolysis was developed using Pronase E. It yielded a hydrolysis rate of 97 % calculated via phenylalanine release. A recovery rate of 2-S-cysteinyl caffeic acid (2-CCA) of 64 % after hydrolysis was achieved when buffer systems containing borate anions were used. The formation of 2-CCA in bovine serum albumin (BSA) incubated with CA was shown.