Abstract: A sensitive, specific and rapid RP-HPLC method was developed for simultaneously determining a synthetic steroid drug danazol (Ⅰ) and its metabolites 2-hydroxymethylethisterone (Ⅱ) and ethisterone (Ⅲ). A column packed with YWG-C18, 10 μm and mobile phase of methanol-water (74:26, V/V) were used to separate C Ⅱ, Ⅲ and chloromadinone (I. S.). In order to get more satisfactory sensitivity of the four compounds, the detection wavelengths were set at 285 nm and 240 nm with maxplotechnique.he chromatographic peaks of Ⅰ and Ⅱ in serum were confirmed by UV spectra and mass spectra. The results showed that the limits of detection using 1 ml serum were 10 ng/ml for Ⅰ, 25.38 ng/ml for Ⅱ and 11.15 ng/ml for Ⅲ. Standard curves were constructed with 1 ml serum contaning Ⅰ (9.855～438.00 ng/ml), Ⅱ (50.75～812.00 ng/ml) and Ⅲ (14.99～428.40 ng/ml). The recoveries of Ⅰ, Ⅱ and Ⅲ from 1 ml serum were 99.04%, 63.17% and 96.08% respectively.The method was used to profile the serum levels of Ⅰ and Ⅱ in female with endometritis receiving 200 mg of danazol b. i. d. The result showed that the concentration of Ⅱ was 5～9 times highter than that of Ⅰ.