A facile high-performance liquid chromatography (HPLC) method for the resolution and quantitative measurement of nine marker substances, the active ingredients in patch preparations of Ru-Yi-Jin-Huang-San, was established using gradient elution in the reversed-phase mode. These marker substances included berberine (Phellodendri Cortex), curcumin (Curcumae Rhizoma), imperatorin (Angelicae Dahuricae Radix), magnolol (Magnoliae Cortex), hesperidin (Citri Leiocarpae Exocarpium), glycyrrhizin (Glycyrrhizae Radix), and emodin, sennoside A, sennoside B (Rhei Rhizoma). The ingredients in the water-based and oil-based patches of the formula from different manufactures were also analyzed for quality evaluation. Extracted samples were analyzed by HPLC using a reversed-phase column (Inertsil 5 ODS-2, 4.6-mm I.D. ×250 mm) at 30°C and eluted with a mixture of 20 and 70% acetonitrile aqueous solution in gradient manner at a flow rate of 1.0 ml/min. The detection wavelength varied with time as follows: 275 nm, 0–72 min; 250 nm, 72–105 min; 220 nm, 105–145 min. Relative coefficients of variations of intra- and interday analysis were less than 5%. All the recoveries were 93.30–113.63%. This method could be applied for the simultaneous determination of nine marker substances in Ru-Yi-Jin-Huang-San.