Abstract: A method of HPLC-MS/MS for the determination of 3 glycopeptide antibiotics,i.e. vancomycin,norvancomycin and teicoplanin in milk was proposed. The sample was extracted with the mixture of acetonitrile and 10% (φ) acetocaustin (6+4). At the same time,(NH4)2SO4 was used for protein precipitation,and the sample solution were cleaned up with HLB solid phase extraction column. XBridge C18 column was used as stationary phase,and the mixture of acetonitrile and acetic acid (0.3+99.7) solution mixed in different ratio was used as mobile phase in gradient elution. ESI+ and multi-reactions monitor were adopted in MS/MS. Linear relationships between values of peak area and mass fraction of 3 compounds were kept in the same range of 5.00-500 μg·kg-1,with detection limits (3S/N) in the range of 1.0-2.0 μg·kg-1. Tests for recovery were made by standard addition method at the concentration levels of 5.00,10.0,20.0 μg·kg-1,giving values of recovery and RSD′s (n=6) in the ranges of 70.0%-83.5% and 4.2%-11%,respectively.