Synthesis of oligopeptides is generally carried out in aqueous or bi-phasic reaction media; use of monophasic reaction media is quite limited. In this study, homo-oligopeptides of lysine (Lys), glycine (Gly), methionine (Met) and tyrosine (Tyr) were synthesized through papain-catalyzed reaction in monophasic systems consisting of acetonitrile/ water. Reaction conditions were optimized for peptide bond formation and to minimize enzyme denaturation. Such media are especially attractive because they can minimize the secondary and reverse hydrolysis of the acyl complex and the oligopeptide, respectively. The synthesized oligopeptides were purified and characterized by reversed phase liquid chromatography (RPLC) and Electrospray ionization mass spectrometry (ESI-MS). The yields of oligopeptides were approximately 80% for all amino acids. The stereo specificity of papain in acetonitrile/water media was also investigated. The separated enantiomers of methionine were characterized using Chiral High-Pressure Liquid Chromatography (HPLC). The results indicate the L-specificity of papain is maintained in monophasic media.