The search for novel peptide substrates for proline-specific peptidases has been hampered by a lack of suitable assays. Existing technologies are often too expensive, labor-intensive or require derivatization and separation of the reaction products. The purpose of this study was to validate the use of isothermal titration calorimetry (ITC) to study enzyme kinetics, in comparison to more conventional methods (e.g. MALDI-TOF mass spectrometry, quantitation of released C-terminal Phe using RP-HPLC). The study led to the discovery of a novel substrate for prolyl carboxypeptidase, an enzyme involved in body weight regulation.