The literature contains several papers describing the kinetic mechanism of the optical resolution of N-acetylo-DL-amino acids catalyzed by L-aminoacylase. Most authors propose ar eversible Michaelis-Menten kinetic reaction scheme inhibited by substrate and products. Such studies are mostly based on initial rate measurements. In this paper an alternative method is presented to determine both the reaction scheme and the value of the involved constants. The method is based on measuring product concentration for different initial substrate concentrations up until the time when equilibrium is reached and on the numerical integration of the late equation. The optical resolution of N-acetyl-DL-phenylalanine and N-acetyl-DL-valine catalyzed by L-aminoacylase were used as model systems to check the validity of the proposed method, and a slightly substrate-inhibited reversible Michaelis-Menten reaction scheme was demonstrated. In disagreement with other previously published studies, no product inhibition was detected Previous experiments were performed to determine the most suitable enzyme concentration and the optimal concentration of the activator Co2+.