Abstract: To establish the domestic standard method, a high performance liquid chromatography (HPLC) assay was developed for ethinylstradiol determination in three key types of feeds (complete formula feeds, concentrated feeds, and vitamin premixes). In this assay, ethinylstradiol was extracted from feeds by acetonitrile-0.025% phosphate (1:1, v/v) solution, then the extraction solutions were purified by liquid-liquid partitioning, and further purified by the reverse phase extraction C-18 cartridge, and later, ethinylstradiol in the purified solutions was determined by reverse phase HPLC coupling fluorescence detector. The results showed that the linear ranges measured by this assay were 0.05-5 mg/L ethinylstradiol, and the linear correlation coefficient was 0.9992. The LOD of this assay for feeds and vitamin premixes was 0.04 mg/kg and 0.06 mg/kg, respectively. The LOQ was 0.1 mg/kg for feeds and 0.2 mg/kg for vitamin premixes. The average recoveries in complete formula feed at three spiked level of 0.05, 0.25, 0.5 mg/kg were between 83% and 87%,with RSD ranged from 5.9% to 7.0%. The average recoveries in three key types of feeds were 81%-86%, and the relative standard deviations (RSDs) of intra-lab and inter-lab were 3.3%-9.6% and 5.7%-6.6%, respectively. The estalished method was environment-friendly, sensitive, and convenient for fast determination of ethinylestradiol in different types of feeds.