A facile HPLC method for the reduction and quantitative measurement of ten marker substances, the active ingredients in formula preparation of Wan-Yin-Gau, was established under the gradient elution in the reversed-phase mode. These marker substances included cinnamic acid,cinnamaldehyde (Cinnamomi Cortex), isoimperatorin (Angelicae Dahuricae Radix and Notopterygii Rhizoma), ferulic dcid (Angelicae Sinensis Radix), paeoniflorin (Paeoniae Radix), glycyrrhizin (Glycyrrhizae Radix), harpagoside (Scrophulariae Radix), emodin, sennoside A, and sennoside B (Rhei Rhizoma). The ingredients in the water-based and oil-based patches from different manufactures were also analyzed for quality evaluation. Extracted samples were analyzed with reverse-phase column (Inertsil 5 ODS 2, 4.6 i.d. × 250 mm) at 30°C, eluted with a mixture of 20% and 70% acetonitrile aqueous solution in gradient manner at a flow rate of 1.0 mL/min, and detected at 250 nm. Relative coefficients of variation of intra- and inter-day analysis were less than 5%. All the recoveries were 96.29~103.46%. This method could be applied for the simultaneous determination of ten marker substances in “Wan-Yin-Gau”.