A method using the high performance liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) to identify trichlorfon, endosulfan and colchicine was described in this study. Data acquisition of the protonated precursor molecular ions (parent ions) and the related product ions (fragment ions or daughter ions) for two pesticides and one drug banned in seafood in the EU and US were performed by the scan mode with a scan range of 50 to 550 m/z or by daughter ion scan mode or in the selected ion monitoring mode. Quantification was performed by multiple reaction monitoring of the molecular ions and the related product ions for each drugs. All parameters of spectra acquisition with LC/ESI-MS/MS for each ion pairs from above three drugs were optimized according to the s/n ratio. The separation of drugs in the high performance liquid chromatography using a gradient elution on a C18 reversed phase column. The retention times for trichlorfon, colchicine and endosulfan were 6.3, 6.5 and 6.9 minutes, respectively. And their detection limits (s/n=10) were under 1, 0.1 and 10 ppb, respectively. The withdrawal time of oxolinic acid, based on 0.5-ppm tolerance residual level, in grouper after single oral administrationat at a dose of 40 mg/kg b.w. at 23.5 and 30oC, respectively, were 10.3 and 8.9 days. Therefore, withdrawal time should be reduced 5 hours when water temperature increase 1oC.