Optically pure 2-chloromandelic acid (ClMA) is a very important chiral drug intermediate for synthesis of (S)-clopidogrel, belonging to the platelet aggregation inhibitor. Enantioselective resolution of (R,S)-2-chloromandelic acid was carried out in organic solvent through irreversible transesterification catalyzed by lipase AK with vinyl acetate acting as the acyl donor. Effects of various conditions on enantioselectivity and activity of lipase were investigated, including organic solvents, temperature, water content, substrate ratio, enzyme loading, and reaction time. Based on homogeneous reaction and Ping-Pong bi-bi mechanism, a quantitative model was constructed to simulate and optimize the reaction process. Under the optimal conditions, excellent results were obtained with high conversion of (R)-2-ClMA (c R , ≥98.85%) and large enantiomeric excess of substrate (ee s , ≥98.15%). There is a good agreement between predicted values and experiment data, which indicates that the established method is a powerful tool for optimization of the enantioselective transesterification process for enantiomers separation.