Separation and quantitative determination of 20-hydroxyecdysone from Achyranthis Radix has been conducted by using HPLC method. 20-hydroxyecdysone in a methanol extract from the raw drug was separated on a reverse phase column using a $CH_3CN-H_2O;(18:82)$ solvent system and the average content is $0.0931{pm}0.0048%$. For the preparation of authentic standard, we isolated 20-hydroxyecdysone from the roots of Achyranthes fauriei by $SiO_2$ and RP-18 column chromatography.