N-Acetyl-phenylalanine-glycinamide (N-Ac-Phe-Gly-NH(2)), a type of dipeptide derivative, was synthesized from N-acetyl phenylalanine ethyl ester and glycinamide and catalyzed by alpha-chymotrypsin, a protease, in a biphasic system. Response surface methodology with a four-factor, five-level central composite rotatable design was employed to evaluate the effects of selected parameters that included incubation time, reaction temperature, enzyme activity, and pH level on the yield of the dipeptide derivative. The results indicated that pH significantly affected the yield of N-Ac-Phe-Gly-NH(2). In a ridge max analysis, the optimum condition for this synthesis included an incubation time of 30.9 min, a reaction temperature of 35.8 degrees C, an enzyme activity of 159.2 U, and a pH of 8.98. The predicted and the actual (experimental) yields were 98.0 and 95.1%, respectively.