Abstract: AIM: To set up an assay method for the determination of ecdysterone (EDS) in the root of Achyranthes bidentata Bl. by RP-HPLC. METHODS: The EDS was separated on a Hypersil-ODS column (200 mm×4.6 mm ID, 5 μm), using acetonitrile — water (1.0∶5.4) as the mobile phase and detected at 243 nm. The flow rate was 1.0 mL.min^-1. Phenol was used as an internal standard. RESULTS: The calibration curve was in good linearity over the range of 28.04～280.40 μg (γ=0.9997). The average recovery was 95.2%. CONCLUSION: The method is simple, fast, sensitive and is suitable for quantitative analysis of EDS in the root of Achyranthes bidentata Bl.