In drug development, the combinatorial synthesis of drug libraries is common use, therefore efficient tools for the characterization of drug candidates and the extent of interaction between a drug and its target protein is a central question of analytical interest. While biological activity is tested today by enzyme assays, MS techniques attract more and more attention as an alternative for a rapid comparison of drug target interactions CE enables the separation of proteins and drug enzyme complexes preserving their physiological activity in aqueous media By hyphenating CE with ESI-MS/MS, the binding strength of enzyme inhibitors can be deduced from MS/MS experiments, which selectively release the inhibitor from the drug target complex after CID In this study, a-chymotrypsin (CT), a serine protease, was chosen as a model compound. Chymostatin is a naturally occurring peptide aldehyde binding to CT through a hemiacetal bond and electrostatic interaction. First, a CE separation was developed, which allows the analysis of alpha-CT and a chymotrypsin chymostatin complex under MS-compatible conditions The use of neutral-coated CE capillaries was mandatory to reduce analyte wall interactions. ESI-quadrupole ion trap-MS was worked out to demonstrate the selective drug release after CID Fragmentation of the drug enzyme complex was monitored in dependence from the excitation energy in the ion trap, leading to the V-50 voltage that enables 50% complex fragmentation as a reference value for chymotrypsin chymostatin complex. A stable CE-ESI-MS/MS setup was established, which preserves the drug enzyme complexes during ionization-desolvation processes With this optimized setup, different CT inhibitors could be investigated and compared