A novel inhibition assay by capillary electrophoresis/dynamic frontal analysis (CE/DFA) is proposed. When substrate p-nitrophenyl phosphate and inhibitor theophylline were tandemly introduced into a capillary containing alkaline phosphatase enzyme, two plateau signals were detected in the electropherogram. A higher plateau is based on the CE/DFAwithout inhibition, and a suppressed plateau is formed while the substrate zone passes through the inhibitor zone. Inhibition constant was successfully determined through the two plateau heights.