The permeation experiments Of L-phenylalanine (L-Phe) and L-aspartic acid (L-Asp) aqueous solutions were carried out with two commercial nanofiltration (NF) membranes, ESNA2 membrane and ES20 membrane. The rejections to L-Phe and L-Asp by ESNA2 membrane are about 0 and 90%, respectively, at the pH value ranging from 4 to 9, while the rejections to L-Phe and L-Asp by ES20 membrane are almost 100% irrespective of pH value. The results show that these two NF membranes are possible to concentrate and separate L-Phe and L-Asp effectively by choosing proper condition such as the pH value of solution. Based on the experimental results, a simple batch-wise NF process system was design for concentration and diafiltration of virtual L-Phe enzymatic reaction solution with Some L-Asp. The simulation results show that it is feasible to separate L-Phe and L-Asp in viewpoint of technology.