Isomerases involved in the metabolism of D/L-amino acids represent promising therapeutic targets for treatment of disease. Herein, we report a tunable platform for the assessment of enzymatic kinetics involving amino acid isomerization by CE that offers improved selectivity and sensitivity over traditional methods. Enzyme activity and competition assays were evaluated for various hydroxyproline diastereoisomers, proline enantiomers and their structural analogs using 4-hydroxyproline-2-epimerase as a model system. In this work, pyrrole 2-carboxylic acid was found to be a selective inhibitor of 4-hydroxyproline-2-epimerase with a half-maximal inhibition concentration of (2.3 +/- 0.1) mM. Reliable methods for unambiguous characterization of amino acid isomerases are required for the screening of novel inhibitors with epimerase and/or racemase activity.