Abstract: A detection method for the determination of 20 kinds of cephalosporins in animal derived food was established with multiple stable isotope internal standards by liquid chromatography-tandem mass spectrometry. Cephalosporins were extracted from samples with methanol, and purified with octadecylsilane sorbent. An Ascentis C₈ column was used for analyzing with 0.1% formic acid aqueous solution and acetonitrile as mobile phases. Analysis of cephalosporins were respectively performed on the positive and negative scan mode by multiple reactions monitoring. Results showed that the linear range of the methods was 5~100 μg/L for 17 kinds of cephalosporins with highly responsive, and 25~500 μg/L for 3 kinds of cephalosporins with lowly responsive. The determination coefficients of the linear equations ranged from 0.9913 to 0.9998. The limit of detection for 20 kinds of cephalosporins ranged from 1.0 to 20.0 μg/kg, and the limit of quantification ranged from 3.0 to 60.0 μg/kg in animal derived sample. The average recovery rate of 20 cephalosporins ranged from 78.08% to 115.47%, and the relative standard deviation ranged from 3.07% to 12.44%. The validated method was simple and accurate. It was suitable for the determination of 20 cephalosporins in animal food.