We developed a simultaneous analysis method using high-performance liquid chromatography coupled with diode-array detector (HPLC-DAD) for six principal compounds (atractylenolide III, alisol A, alisol B, paeoniflorin, ferulic acid and (Z)-ligustilide) in a traditional Japanese (Kampo) medicine, tokishakuyakusan (TSS). The HPLC separation was conducted on a reversed-phase TSK-gel ODS-80TS column (4.6 i.d. × 250 mm, 5 µm) at 40°C with a 0.1% phosphoric acid-acetonitrile gradient system. The DAD detection wavelength was set at 205, 232 and 330 nm. Calibration curves for the compounds showed linear regressions with correlation coefficients of >0.999. The intra- and inter-day precision (i.e., the relative standard deviation) were in the range of 0.50-1.55 and 0.70-1.80%, respectively. The average recovery yields of the compounds ranged from 98.3 to 103%. The present results will contribute to shorter analysis times with less organic solvent compared with the individual analysis of each compound for the evaluation of TSS. The application of the established method to TSS will also provide helpful information for the further pharmacological and clinical studies.