Three strong anion-exchange membranes (CIM QA, Q100 and HiTrap Q) were investigated for the separation of the major proteins, which were contained in whey, such as α-Lactalbumin, BSA and β-Lactoglobulin. Experiments were performed to determine the optimum mobile phase composition for separating the whey proteins using the standard chemicals of the proteins. The mobile phase was buffer A (20 mM piperazine-HCl pH 6.4) and buffer B (buffer A+1 M NaCl) and the linear gradient elution changes of salt concentration were applied. The standard chemicals of the proteins were used to investigate the optimal mobile phase compositions with the three anion-exchange membranes. From the experimental results, it was found that HiTrap Q was the most effective in separating whey proteins.