Abstract: Objective:A ultra-high pressure liquid chromatograph-tandem mass spectrometry (UPLC-MS/MS) was established for determination of desfuroylceftiofur residues in raw milk. Methods:T3 column was used with a mixed solution of 0.02% formic acid-acetonitrile as mobile phase. The cefliofur in bovine raw milk was decomposed by dithioerythritol to produce desfuroylceftiofur (DFC) , which was further stabilized by derivatization to desfuroylceftiofur acetamide (DCA) using iodoacetamide, then the samples were concentrated and purified by HLB SPE cartridge and determined by UPLC-MS/MS.Results:The quantitative ion was m/z 430.12>125.79.The qualitative ion was m/z 430.12>241.04. The recovery were 98.6%~102.2%, the relative standard deviations (RSD) were 2.51%~3.87%, the minimum detectable values were 0.45ng/mL. Conclusion:This method could be used for the routine analysis of desfuroylceftiofur residues in raw milk.