Abstract: A method based on high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) had been developed for simultaneous determination of 6 aminoglycoside and 3 tetracycline antibiotics residues in crayfish. The antibiotics were ultrasonic extracted from crayfish muscles with 5% trichloroacetic acid-phosphate buffer solution,purified by hydrophilic-lipophilic balance(HLB) cartridge and separated by C_(18) chromatographic column. The pre-treatment method,chromatographic and MS/MS parameters were optimized.The limits of detection for streptomycin,dihydrostreptomycin,tetracycline,chlortetracycline and oxytetracy-cline were 5 μg/kg,respectively,and for gentamycin,kanamycin,neomycin andtobramycin 50 μg/kg,respectively.The recoveries of 9 antibiotics residues at three spike levels ranged from 66.1% to 107.9% with the relative standards deviations of 0.7%~9.7%. The established method was accurate and effective for confirmation and quantification of 9 different aminoglycoside and tetracycline antibiotics residues in crayfish.