Abstract: A simple, rapid and specific analytical methods for the determination of vancomycin and norvancomycin in milk had been developed based on liquid chromatography tandem mass spectrometry in a multiple reaction monitoring mode. Milk samples were extracted with 0.1% trifluoroacetic acid in water. Antibiotics in the extract were separated on a ZORBAX SB-C18 column using a gradient elution program of 0.1% formic acid aqueous solution and acetonitrile. Limits of detection for vancomycin and norvancomycin were 5μg/kg. Mean recoveries were 72.5%～87.2%, and the relative standard deviation (RSD) was less than 10%.