Abstract: OBJECTIVE To establish a method of the simultaneous determination of 4 components in Belladonna liquid extract, provide the basis for its quality evaluation by combing with TLC. METHODS Adopting HPLC and using Agilent Kromesil 100-5 C₁₈ chromatographic column(4.6 mm×250 mm, 5 μm), the mobile phase was a mixture of acetonitrile and 0.05% formic acid for gradient elution. The flow rate was 1.0 mL·min^-1. The temperature of column was 30℃. Atropine sulfate was identified with TLC method. RESULTS Four active ingredients had good linear relationships:scopolin was in the range of 0.065 76-0.657 6 μg(r=1.000 0), kaempferol-3-O-galactosyl(6→1)-rhamnosyl-7-O-glucoside was in the range of 0.082 16-0.821 6 μg(r=0.999 9), scopoletin was in the range of 0.049 84-0.498 4 μg(r=1.000 0) and rutin was in the range of 0.010 00-0.099 98 μg(r=0.999 9). The average recoveries of scopolin, kaempferol-3-O-galactosyl(6→1)-rhamnosyl-7-O-glucoside, scopoletin and rutin were 100.6%, 99.8%, 99.9% and 97.9%. And their RSDs were 1.4%, 1.6%, 1.4% and 1.5%. The contents of some ingredients were low and atropine sulfate was detected in some samples. CONCLUSION This method has high sensitivity, it is accurate and suitable for the quality control of Belladonna liquid extract.