Abstract: OBJECTIVE To establish a specific HPLC chromatogram of belladonna tincture and a method for determination of scopoletin. METHODS A Welch XB-C₁₈ column (250 mm×4.6 mm, 5 μm) was adopted, methanol-0.05% phosphoric acid was used as the mobile phase for gradient elution, the flow rate was 1.0 mL·min^-1, the column temperature was 30 ℃, and the wavelength was set at 344 nm. RESULTS The chromatographic fingerprint was completed with 6 recognizable peaks, of which 3 peaks kaempferol-3-O-β-D (6-O-α-L-rhamnogalactoside)-7-O-β-D-glucoside, scopoletin and rutin were determined. The linear range of scopoletin was 11.18-670.5 ng (r=1.000 0), the average recovery was 104.2%. CONCLUSION The combination of specific chromatograms and determination of scopoletin is suitable for the quality control of belladonna tincture.