Abstract: 【Objective】 An analytical method was developed for the determination of amoxicillin residues in milk by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).【Method】 A two-gram milk sample was deproteinized by ethanol．The supernatant was transferred into a pear-shaped bottle to be evaporated to about 0.5 mL，and the residue was dissolved with ammonium acetate solution．The sample was determined by HPLC MS/MS after the purification．The chromatographic separation was achieved on a Luna 5 μm C₁₈ column using 0.1% formic acid in water and acetonitrile as mobile phases with gradient elution．The identification of amoxicillin was carried out by MS/MS equipped with electrospray ionization in positive scanning and multiple reaction monitoring (MRM) modes．【Result and conclusion】 Matrix-matched calibration standard was used for the quantification．The calibration curve showed perfect linearity in the range of 1-400 μg·L^-1, with the correlation coefficient being more than 0.999．The limit of detection (LOD，S/N≥3) was 1 μg·kg^-1 in milk，and the limit of quantification (LOQ，S/N≥10) was 2 μg·kg^-1．The mean recoveries varied from 75.6% to 91.0% at the four spiked levels of LOQ，1/2MRL (the maximum residue limit: 5 μg·kg^-1)， MRL(10 μg·kg^-1)，and 2MRL(20 μg·kg^-1) with the relative standard deviations of 1.6%-10.2%．