Abstract: A method combining SPE and Ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) was developed for the simultaneous determination of nine polypeptide antibiotics in animal tissues. The samples were extracted by methanol and 0.1 mol/L hydrochloric acid (7:3, v/v), then cleaned with Alumina-A and n-hexane, and finally purified by hydrophilic-lipophilic balance (hydrophilic-lipophilic balance, HLB) cartridge. The separations were performed on a C₈ column (100A, 150 mm×2 mm, 3 µm) using a mobile phase gradient with 0.2% formic acid and acetonitrile. Electrospray positive ionization and multi-reaction monitoring (MRM) mode were used for detection. The calibration curves for vancomycin, norvancomycin, enramycin A, enramycin B, and teicoplanin, (concentration range 2~200 μg/L), colistin A, colistin B and bacitracin A (concentration range 5~500 μg/L), virginiamycin M1 (concentration range 0.1~20 μg/L) were linear with correlation coefficients more than 0.99. The limits of quantification (LOQ, S/N=10) of 9 kinds of polypeptide antibiotics were varied from 1 µg/kg to 20 µg/kg, the detection limit of the method was 0.3 µg/kg to 6 µg/kg. The recovery experiments were performed with chicken, pork, liver and kidney samples spiked in the range of 1~200 μg/kg. The average recoveries ranged from 74.2% and 96.3% with the relative standard deviations (RSDs) of 4.3%~14.6% (n=6). The method is simple and rapid with high sensitivity and good reproducibility. Furthermore, this method is effective for the safety monitoring of polypeptide antibiotic residues in animal tissues.