An ultra performance liquid chromatography-tandem mass spectrometric method（UPLC-MS/MS） was established for the simultaneous determination of amoxicillin and polymyxin E residues in animal derived food.Mass spectrometric study on amoxicillin and polymyxin E was performed,and UPLC-MS/MS conditions were optimized.Samples were extracted with trichloroacetic acid solution.The protein was precipitated with lead acetate and the fat was removed with hexane.The other impurities were removed with HLB solid phase extraction cartridge.After concentration,the analytes were detected by UPLC-MS/MS and quantified with the matrix-matched standard.The limits of detection（LOD,S/N＞3）for amoxicillin and polymyxin E were 6 μg/kg and 15 μg/kg，and the limits of quantitation （LOQ,S/N＞10） were 20 μg/kg and 50 μg/kg,respectively.The average recoveries at three spiked concentrations in muscle,liver,kidney and fat were 76%-96% for amoxicillin and 74%-92% for polymyxin E with RSDs less than 10%.Furthermore,this method was applied in the determination of amoxicillin and polymyxin E residues in swine muscle,liver,kidney and fat for elimination study,and the withdraw times were caculated on the basis of the result.The result indicated that the method was simple,rebust and was suitable for the simultaneous quantitative detection of amoxicillin and polymyxin E residues in animal-derived food.