Abstract A potentiometric method utilizing a leucine-selective membrane electrode has been developed for the assay of proteolytic enzyme. Subtilisin Carlsberg was used for the enzymatic digestion of casein in solution. Responses on the reaction media were linear on a semilog plot for active enzyme concentrations ranging from 0.6–60 μg/ml. The system uses commercially available components as well as simplified methods of solution handling without precipitation or filtration steps. This method offers significant advantages in terms of simplicity, speed, and cost over the traditional techniques.