In reversed-phase high performance liquid chromatography (RP-HPLC), the mobile phase condition for separating eight isoflavones (daidzin, glycitin, genistin, 6'-o-acetyl daidzin, 6'-o-malonyl genistin, daidzein, glycitein and genistein) was optimized using the HCI (High-Purity Separation Laboratory, Department of Chemical Engineering, Inha University) program software. The optimum composition of mobile phase for the separation of the eight isoflavones was obtained. The elution profiles were calculated by the plate theory based on the equations of retention factor, In k=A+BF+CF2, where F was the volume percentage of acetonitrile with 0.1% acetic acid (AA). The first mobile phase composition was water with 0.1% AA/acetonitrile with 0.1% AA (88%/12%, by volume), followed at 9min later by the second composition of mobile phase which was step-changed to 85%/15%, at 19min by the third composition which was step-changed to 73%/27%, at 30min when it was changed to 65%/35% and finally it was maintained in isocratic mode to the end of the run time at 50min. Although, using step gradient mode to separate the isoflavones, the calculated and experimented data were not achieved very good agreement, we could estimate the closed retention time before experiment. And the agreement between the experimental data and the calculated values was relatively good using isocratic separation for eight isoflavones, but the retention time is very long.