Abstract: AIM　To study the activity of ecdysterone from Achyranthes bidentata Bl. (AB) promoting proliferation of osteoblast-like (OB-like) UMR106 cells and to determine its content in AB by HPLC method. METHODS　Ecdysterone isolated from AB was cultured with OB-like cells UMR106 together in vitro and the proliferation of OB-like cells was determined by MTT assay. The chromatographic conditions for determining ecdysterone included an ODS column (250 mm×4.6 mm, 5 μm), a mobile phase consisting of a mixture of water-acetontrile-tetrahydrofuran (86∶11∶3), detection wavelength of 243 nm, and column temperature of 27℃. Phenacetin was used as the internal standard. RESULTS　The ecdysterone from AB had significant activity promoting proliferation of OB-like cells, the proliferation was promoted by 41% (n=3). The average recovery of ecdysterone was 96.2% (RSD=2.1%), the calibration was linear in the range of 30～300 μg.mL^-1 (γ=0.9998). CONCLUSION　Ecdysterone was screened quickly by cultivating with OB-like cells together in vitro. The HPLC method is accurate, fast and reproducible for the determination of ecdysterone in AB.