Abstract: The sample (0.100 0 g) of tobacco licorice extract was extracted ultrasonically for 30 min with 10 mL of 80% (φ) methanol solution, and then the above mixture was stood for 30 min. The supernatant was taken and filtered on 0.45 μm filtering membrane. HPLC was applied to the simultaneous determination of 14 active constituents in the filtrate. Diamonsil Plus C₁₈ chromatographic column was used as separated column with mixtures of 0.05% (φ) phosphoric acid solution and acetonitrile with different mixing ratios as mobile phase in the gradient elution, and photodiode array detector was used for determination. The extraction conditions were optimized by single factor tests and L9(3³) orthogonal test. Linear relationships between values of peak area and mass concentration of the 14 active constituents were kept in definite ranges. Detection limits (3s) of the method ranged from 0.02 to 0.12 mg·L^-1. Tests for recovery were made by standard addition method at 3 concentration levels, giving values of recovery and RSDs (n=5) of determination in the ranges of 91.7%-143% and 1.2%-6.0% respectively.