As a potential anticancer agent, the isoflavones contained in soybeans were considered for separation by reversed-phase high performance liquid chromatography (RP-HPLC). The mobile phases were the ternary system of water/acetonitrile/acetic acid. In this work, the four different columns of μ-Bondapak (0.39 × 30 cm, 10 μm), and the other three empty stainless steel columns (0.39 × 30, 1 × 25, 1″ × 25 cm) packed with 15 μm C18packings were used. The experimental variables were the gradient conditions and mobile phase composition. The increased number of gradient steps enabled the separation of isoflavones in the larger columns. The separation condition of 1″ × 25 cm column was determined by experiments of the systematic sequence of the smaller columns. For the largest column, 2 mL of sample was separated with the three gradient steps at 18 mL/min of mobile phase flow rate. The isoflavones of daidzein, glycitin, genistin, daidzin, and glycitein were identified by LC-MS with the purified components.