Abstract: 【Objective】 This study was conducted to establish a high-performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS） method in order to optimize the detection technology and improve accuracy and efficiency of detecting antimicrobial drug residues in mutton and its by-products,specifically enrofloxacin,ciprofloxacin,ofloxacin,gentamicin,tilmicosin,and tylosin in mutton and its byproducts.【Method】 The study compared the extraction efficiency of 0. 1% formic acid acetonitrile and 0. 1% formic acid methanol. The purification effects of HLB and C18 solid-phase extraction columns on the six target antimicrobials were compared against national standards for detecting fluoroquinolones,aminoglycosides,and macrolides in animal food. Various eluents,including 0. 1% formic acid water,acetonitrile,0. 1% formic acid acetonitrile,methanol,and 0. 1% formic acid methanol,were used to elute the extracted and purified samples,and their extraction and elution effects were compared. The established method was applied to the detection of actual samples and compared with the national standard.【Result】 The results demonstrated that accurate information regarding enrofloxacin,ciprofloxacin,ofloxacin,gentamicin,tilmicosin,and tylosin could be captured by mass spectrometry using 0. 1% formic acid acetonitrile extraction for 30 minutes,followed by washing with 0. 1% formic acid methanol. The regression coefficients（R2） of the standard curves were all greater than 0. 99 within the limited concentration range. The recovery rates ranged from 73. 3% to 114. 0%,and the relative standard deviations ranged from 1. 01% to 13. 01% at concentrations of 5,20,and 50 μg/kg. The detection limits（LOD） and quantitative limits（LOQ） of the six drugs in three types of edible sheep tissues were found to be in the range of 0. 05～1. 2 μg/kg and 0. 16～3. 8 μg/kg,respectively,which were lower than the detection limits stipulated in current national standards.Single-factor analysis of variance indicated no significant difference between the established method and the national standard method（P＞0. 05）.【Conclusion】A sensitive,accurate,and efficient HPLC-MS/MS method was established for the detection of enrofloxacin,ciprofloxacin,ofloxacin,gentamicin,tilmicosin,and tylosin residues in edible lamb tissues which could serve as a technical reference for monitoring,screening,and accurate identification of antimicrobial residues in mutton and its by-products.