Abstract: A method was developed for the determination of ethinylestradiol residue in powdered milk with HPLC. Samples were extracted with acetonitrile，contrifugated，the extract was filtrated with 0.45 μm filter membrane after being concentrated，freezed and degreased，then carried out HPLC analysis. Extraction solution，extraction method and HPLC condition were optimized. Used acetonitrile-water （50∶50）as the mobile phase，flowing speed was 1 mL/min，column temperature was 30℃，detective wavelength was 224 nm. The detection limit of the method was 16 μg/kg， the recovery was 86.5%-97.0%，the related coefficient of the working curve was 0.999 0，the relative standard deviation was 0.95%-2.01%（n = 3） . The method is rapid，sensitive，good seperation effect，simple，no disturbance from impurity，can meet the request of etinylestradiol dectection in powdered milk.

Key words: HPLC, detection on powdered milk, ethinylestradiol, food security