Abstract: An ultra performance liquid chromatography tandem mass spectrometry(UPLC-MS-MS) method had been established for the simultaneous determination of eight polypeptide antibiotics in animal derived food.Test sample was extracted by methanol-water-formic acid(40∶60∶0.1,v/v/v),followed by cleaning up method based on solid phase extraction(SPE). Extracts were separated on a C18 column using a gradient elution of0.1% formic acid water solution and 0.1% formic acid acetonitrile solution. Analytes were detected by ultra-perform liquid chromatography tandem mass spectrometry in positive ion mode with selected reaction monitoring(SRM). The results showed that the limits of detection(LOD) of 8 polypeptide antibiotics were 0.1~10 μg/kg.The calibration curves for norvancomycin and vancomycin(concentration range 20~1000 μg/L),polymyxin B and E(concentration range 40 ~1000 μg/L),bacitracin A(concentration range 10 ~1000 μg/L),bacitracin B(concentration range 6~614 μg/L),virginiamycin M1 and S1(concentration range 1~100 μg/L) were linear with correlation coefficients more than 0.999. The recoveries were in the range of 61.0% ~99.6%. The relative standard deviations(RSDs) ranged from 1.2%~9.8%. The method was simple and rapid with high sensitivity and good reproducibility,and was appropriate for the identification and quantification of polypeptide antibiotics residue in animal derived food.