To develop a quantification method for chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FF) in fish muscle by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).MethodsCoupled with internal standard of d5-chloramphenicol, samples were extracted with ethyl acetate,then defatted by hexane. The extraction was separated by gradient elution with methanol-water in ACQUITY BEH C₁₈ column. The analytes were simultaneously quantified by the isotope internal standard under the negative ion mode and multiple reactions monitoring mode. ResultsThe limits of detection for CAP,TAP and FF were 0.01,0.03 μg/kg and 0.03 μg/kg, and average recoveries 83.2%-96.5%,76.8%-93.8% and 79.6%-95.0% separately. The relative standard deviation varied between 4.1%-8.8%.ConclusionThe method was selective,sensitive,accurate and suitable for determination of trace three chloramphicols analysis in fish muscle.