Abstract: α-and β-Isomers of santonin were separated by means of HPLC, with Lichrosorb RP-8 reversed-phase column, and methanol—water (40:60) as mobile phase. The effect of temperature and composition of the mobile phase on the chromatographic process was investigated. Results showed that the capacity factors of α-and β-isomers, and the resolution (Rs) decrease as the methanol concentration increases, while their log K' values manifest a linear relationship with the logarithms of the percentage concentrations of methanol. Under the present experimental conditions, the symmetry factor of α-santonin is 1.02, number of theoretical plate, 16,000/M, resolution, Rs 1.57, thus the isomers can be well separated. 4-Amino-antipyrine was chosen as internal standard so that a quantitative analysis by HPLC has been successfully established, as there is a good linearity between the internal standard and samples. The method is simple, convenient and rapid, yielding satisfactory results.