In this study, a novel and simple high performance liquid chromatography with diode array detection (HPLC-DAD) method for the simultaneous qualitative and quantitative determination of 12 bioactive components in Gegen Qinlian pill was developed. The separation was performed on a Kromasil C18 column (250 mm×4.6 ram, 5.0 μm) by gradient elution with acetonitrile and 0.02 mol/L ammonium acetate (containing 0.3% triethylamine, and adjusted pH to 4.3 using 1% glacial acetic acid) as the mobile phase at a flow rate of 0.7 mL/min. Three different detection wavelengths (250, 280, 346 nm) were set at the maximum UV absorption wavelengths of these components. Twelve components (puerarin, daidzin, baicalin, wogonoside, liquiritin, berberine, palmatine, jatrorrhizine, glycyrrhizin, baicalein, wogonin and daidzein) were identified and determined using the developed method. All calibration curves showed good linear regression (r〉0.9995) within tested ranges. The injection precision, intra-day precisions and analysis repeatability were evaluated with the RSD values, which were no more than 0.97%, 1.69% and 1.71%, respectively. The recoveries were ranged from 97.4% to 100.2% with RSD values less than 1.87%. This readily available, low-cost and reliable HPLC-DAD method would improve the quality control of Gegen Qinlian pill.