The action of serine (and cysteine) proteases on peptide esters proceeds, as a generalization, orders of magnitude faster than the corresponding enzymatic hydrolysis of peptide bonds or peptide amides. Esterolysis liberates an alcohol while generating a free carboxyl group on the peptide; the proton produced can be detected by the use of an appropriate indicator. The action of trypsin on benzyloxycarbonylalanylarginine methyl ester was used as a model for the development of a simple microtiter plate assay procedure that takes advantage of the speed of these reactions and the ease of detection afforded by the color change of the indicator. A family of ester substrates of the form benzyloxycarbonylalanyl-X-methyl ester, in which (X) is one of the 20 common amino acids, was synthesized to allow the determination of the primary specificity profiles of serine proteases. Using a 96 -well microtiter plate the specificity profiles of four enzymes with all 20 substrates can be carried out in approximately (4 \mathrm{~h}) per enzyme, including setting up and data processing. The primary substrate preferences of trypsin, chymotrypsin, thrombin, pancreatic elastase, (\alpha)-lytic protease, subtilisin, and proteinase (K) were determined to demonstrate the method and were found to be in good general agreement with reported specificities established by more conventional means. (c) 1994 Academic Press, Inc.