The aqueous solubility of some amino acids is very low which can be improved by dipeptides in case of these molecules. Although tyrosine is one of those amino acids that have a low aqueous solubility, dipeptides on the other side, such as L-glycyl-L-tyrosine (Gly-Tyr), have high aqueous solubility. Gly-Tyr is rapidly degraded into Lglycine and L-tyrosine in the human body and useful as a transfusion material. Here, the PST-01 protease from Pseudomonas aeruginosa PST-01 had high stability in the presence of organic solvents having high tyrosine solubility. Therefore, an efficient synthetic method of a precursor of Gly-Tyr was investigated using the PST-01 protease. In this study, we investigated the reaction conditions of the synthesis of carbobenzoxy-glycyl-L-tyrosine amide (Cbz-Gly-Tyr-NH2), in the presence of organic solvents. As a result, Cbz-Gly-Tyr-NH2 was successfully synthesized using the PST-01 protease in the presence of 60 % (v/v) dimethylsulfoxide at the maximum equilibrium yield of 81.9 %.