In this study, a reverse-phase high performance liquid chromatography (RP-HPLC) method with high separation efficiency, high detection sensitivity and excellent selectivity was successfully developed for the simultaneous determination of the aloe-emodin, rhein, emodin, chrysophanol, and physcion compounds. These optimum method conditions were determined by studying various columns, mobile phases and compositions, flow rates and column temperatures. The successful separation was carried out with a Supelcosil LC-18 column (250 × 4.6 mm, 5 µm) and a gradient program. The procedure was carried out at 20 °C with the flow rate of 1.0 mL/min. and the injection volume of 20 μL utilizing an RP-HPLC method with DAD detector at 225 nm. Deionized water containing 0.5% (v/v) orthophosphoric acid and methanol was used as the mobile phases A and B. This study showed an optimized analytical method can be effectively utilized to the qualitative determination of anthraquinone compounds.