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.