An approach has been developed for predicting the change of the aqueous phase pH due to the partition of amino acid substrates in aqueous-organic biphasic systems. In the model reaction of papain-catalyzed synthesis of N-benzyloxycarbonyl-glycine-L-phenylalanine methyl ester (ZGPM) in an aqueous-ethyl acetate system, the effects of the initial aqueous phase pH and phase volume ratio on the final aqueous phase pH, and the change of the final aqueous phase pH during the course of ZGPM synthesis, are investigated. The results suggest that the approach is useful for selecting optimum reaction conditions for peptide synthesis not only in the aqueous-organic biphasic systems with free or immobilized enzymes but also with an immobilized enzyme in an organic solvent.