Abstract: An HPLC method for the simultaneous determination of residues of tylosin（TYL）,tilmicosin(TIL), chloramphenicol(CAP) and florfenicol(FF) in animal-derived food was developed. The muscle tissue samples were extracted by acetonitrile,then defatted with hexane, and cleaned up on a HLB SPE column. The analytes were separated on a reversed-phase C18column with the methanol-20 mmol/L NaH₂PO₄ solution (pH=3.0,containing 10% methanol), the UV detector was set at λ=275 nm. The standard curves were linear in the range of 0.1～20.0 mg/L for all analytes with correlation coefficients of 0.9970～0.9992.The detection limits for tylosin,tilmicosin, chloramphenicol(CAP), and florfenicol(FF),were 20 μg/kg, 32 μg/kg, 19 μg/kg, 16 μg/kg, respectively. The average recoveries of four analytes ranged from 75% to 87%. The relative standard deviations were between 1.35％～5.41％. The method can meet the requirement for the simultaneously determination of tylosin, tilmicosin, chloramphenicol(CAP), and florfenicol(FF) in beef products.