A method for determining residues of four botanical insecticides oxymatrine, matrine, rotenone, and azadirachtin in fish by liquid chromatographyelectrospray tandem mass spectrometry (LC-MS/MS) is described. The extraction was achieved using acetonitrile, with the addition of sodium chloride to induce a salting out effect before using n-hexane to defat. Chromatographic separation is achieved on Phenomenex Luna C18 column in the mobile phase composition of acetonitrile and 5 mmol/L ammonium acetate buffer consisting formic acid to provide protons for LC-MS/ MS analysis. Accomplishing with the matrix matched calibration curves to compensate for the matrix effect, the quantitative data showed good linear response within the concentration ranges studied. Detection is carried out using positive-ion electrospray tandem mass spectrometry. Calibrations were linear over a working range of 0.5-50 μg/L for oxymatrine, matrine, rotenone, and 2-200 μg/L for azadirachtin. The average recoveries and the relative standard deviation ranged from 88.6-95.7% and 7.58-10.2%, respectively, in spiked fish samples at concentration levels ranging from 0.5 to 10 μg/kg for oxymatrine, matrine, and rotenone and from 2 to 50 μg/kg for azadirachtin. The limits of detection for oxymatrine, matrine, rotenone, and azadirachtin were 0.29, 0.37, 0.21, and 1.4 μg/kg, respectively. The method is accurate, specific, and sensitive for the analysis of the studied botanical insecticides residues in fish samples.