A modified QuEChERS sample preparation combined with high performance liquid chromatography-heated electrospray ionization-tandem mass spectrometric(HPLC-HESI/MS/MS) method was established for the simultaneous determination of niclosamide(NIC)，chloramphenicol(CAP)，thiamphenicol(TAP)，florfenicol(FF) and florfenicol amine(FFA) in plasma，muscle，skirt，liver and kidney of Chinese soft-shelled turtle(Trionyx sinensis).The sample was extracted and purified using a modified QuEChERS method，and ammoniate acetonitrile and octadecyl silane(C18) were chosen as extractant and purification，respectively. The mobile phase comprised of methanol and water，and the flow rate was set at 0.3mL/min.The sample prepared was separated on a Waters Symmetry? C18(2.1mm×100mm，3.5μm) column,and determined in the positive and negative ion multiple reaction monitoring mode through polarity switching between time segments.CAP，TAP，FF and FFA were quantified by internal standard calibration curve method，and NIC was used the external standard matrix-matched standard curve method.Good linearities were observed in the range of 0.3-100μg/L for five analytes，with correlation coefficients not more than 0.9987.The average recoveries of 5 analytes at the spiked level of 1-20μg/kg in plasma，muscle，skirt，liver and kidney of Chinese soft-shelled turtle(Trionyx sinensis) ranged from 77.9% to 105.3% with relative standard deviations(RSD) of 2.7%-10.5%.The detection limits(LOD) for NIC，CAP，TAP，FF and FFA in Chinese soft-shelled turtle tissues were 0.5，0.1，0.5，0.5,0.5μg/kg，and the quantitation limits(LOQ) were 1.0，0.3，1.0，1.0,1.0μg/kg，respectively. The method is simple，accurate，highly sensitive，and is suitable for the simultaneous determination of NIC，CAP，TAP，FF and FFA residues in Chinese soft-shelled turtle tissues.A modified QuEChERS sample preparation combined with high performance liquid chromatography-heated electrospray ionization-tandem mass spectrometric(HPLC-HESI/MS/MS) method was established for the simultaneous determination of niclosamide(NIC)，chloramphenicol(CAP)，thiamphenicol(TAP)，florfenicol(FF) and florfenicol amine(FFA) in plasma，muscle，skirt，liver and kidney of Chinese soft-shelled turtle(Trionyx sinensis).The sample was extracted and purified using a modified QuEChERS method，and ammoniate acetonitrile and octadecyl silane(C18) were chosen as extractant and purification，respectively. The mobile phase comprised of methanol and water，and the flow rate was set at 0.3mL/min.The sample prepared was separated on a Waters Symmetry? C18(2.1mm×100mm，3.5μm) column,and determined in the positive and negative ion multiple reaction monitoring mode through polarity switching between time segments.CAP，TAP，FF and FFA were quantified by internal standard calibration curve method，and NIC was used the external standard matrix-matched standard curve method.Good linearities were observed in the range of 0.3-100μg/L for five analytes，with correlation coefficients not more than 0.9987.The average recoveries of 5 analytes at the spiked level of 1-20μg/kg in plasma，muscle，skirt，liver and kidney of Chinese soft-shelled turtle(Trionyx sinensis) ranged from 77.9% to 105.3% with relative standard deviations(RSD) of 2.7%-10.5%.The detection limits(LOD) for NIC，CAP，TAP，FF and FFA in Chinese soft-shelled turtle tissues were 0.5，0.1，0.5，0.5,0.5μg/kg，and the quantitation limits(LOQ) were 1.0，0.3，1.0，1.0,1.0μg/kg，respectively. The method is simple，accurate，highly sensitive，and is suitable for the simultaneous determination of NIC，CAP，TAP，FF and FFA residues in Chinese soft-shelled turtle tissues.