OBJECTIVE: To establish a method for content determination of umbelliferone and daphnoretin in ethanol extract of Miao medicine Wikstroemia indica simultaneously. METHODS: HPLC method was adopted. The determination was performed on Diamonsil C18 column with mobile phase consisted of acetonitrile-0.2% phosphoric acid solution (gradient elution) at a flow rate of 1.0 mL/min. The detection wavelength was set at 324 nm during 0-15 min and 346 nm during >15-45 min. The column temperature was 30 ℃, and injection volume was 20 μL. RESULTS: The linear range was 0.33-6.60 μg for umbelliferone (r=0.999 7) and 0.21-4.20 μg for daphnoretin (r=0.999 2). The detection limits were 6.60 and 4.20 ng, and the limits of quantification were 18.90 and 12.60 ng, respectively; RSDs of precision, stability (48 h) and reproducibility tests were lower than 2% (n=6 or n=7). The average recoveries were 98.27% (RSD=1.96%, n=9) and 99.98% (RSD=1.84%,n=9), respectively. CONCLUSIONS: The method is simple, accurate, stable and reproducible. It is suitable for content determination of umbelliferone and daphnoretin in Miao medicine W. indica simultaneously.