Abstract: A method was developed for the screening and quantification of 8 steroid hormones including (boldenone, 17-alpha-Methyltestosterone, 19-Nortestosterone, trenbolone, medroxyprogesterone, melengestrol, megestrol-17-acetate, 17-alpha-Hydroxyprogesterone) in feed by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The effects of extraction solvent, protein removal and degreasing conditions on the recovery of 8 steroid hormones were investigated. Finally, acetonitrile was selected as the extraction solvent, protein was removed with trichloroacetic acid and sodium hydroxide, and fat was removed with magnesium chloride and n-hexane. In the chromatographic analysis, the 8 target compounds were separated by Athena C18-WP (2.1×150 mm, 5 μm) column and gradient elution with 5 mmol/L ammonium acetate (1‰ formic acid) and acetonitrile (1‰ formic acid), detected in positive multiple reaction monitoring (MRM) and quantified with internal standards. The results showed that the 8 steroid hormones exhibited good linear relationships in the range of 0~10 μg/L with correlation coefficients (r) of more than 0.9995;the limits of detection (LODs, S/N ≥ 3) were 0.10~0.34 μg/kg and the limits of quantification (LOQs, S/N ≥ 10) were 0.35~0.98 μg/kg. Average recoveries were 70.4%~109% with relative standard deviation of 0.38%~10.3% (n=6). The method was simple, rapid, sensitive and reproducible, and was suitable for rapid screening and detection of 8 steroid hormones in feed.