Abstract: HPLC was applied to the determination of glaucocalyxin A and glaucocalyxin B in isodon japonica. The sample was extracted ultrasonically with methanol, and after filtering through 0.45 μm filtering membrane, the solution was used for HPLC analysis. Hypersil ODS C18 chromatographic column (4.6 mm×200 mm, 5 μm) was used for separation and a mixture of methanol and water (55+45) was used as the mobile phase. Detection with diode array detector at the wavelength of 231 nm was adopted. Linear relationships between values of peak area and mass concentration of glaucocalyxin A and glaucocalyxin B were obtained in the ranges of 0.018-0.575 g·L-1 and 0.009-0.298 g·L-1 respectively. Values of recovery and RSD′s (n=6) found were 102% and 0.084% respectively for glaucocalyxin A, and 103% and 0.071% respectively for glaucocalyxin B.