Optical resolution of racemic mixtures of arginine and alanine was performed by chiral selective nanofiltration membrane. The chiral selective layer of the membrane was prepared by interfacial polymerization of metaphenylenediamine, trimesoyl chloride, and S (-)-2-acetoxypropionyl chloride (S-2-actoxpcl) in situ on the top of polysulfone ultrafiltration membrane. S-2-actoxpcl consists of a chiral carbon atom that has an induced chiral environment in the membrane. The membranes were characterized by FTIR, scanning electron microscopy, and atomic force microscopy to establish a structure-performance relationship. The optical resolution was performed on the membrane testing module and the effect of process parameters was determined. The results indicated that the incorporation of S-2-actoxpcl made membrane chiral selective hence membranes performed optical resolution. The resolution capacity increased by increasing S-2-actoxpcl in polymerizing solution up to 0.03% but any increase beyond 0.03% reduces the resolution capacity. More than 92% enantiomeric excess of D enantiomer was observed in the permeate of the membrane which was prepared from 0.07% trimesoyl chloride and 0.03% S-2-actoxpcl. The membrane prepared without the chain terminator exhibited less volumetric flux but more solute rejection compared to those prepared with the chain terminator. The flux of the membrane increases as the amount of the chain terminator in the reaction increases.