This study aims to develop an analytical method for the simultaneous determination of pethidine and norpethidine in mouse plasma using the liquid chromatographyelectrospray ionization source-tandem mass spectrometry (LCESI-MS-MS) method. After being alkalified, the plasma specimens were extracted by liquidliquid extraction with ethyl acetate. Chromatographic separation was performed on a C-18 column (150 2.1 mm internal diameter, 5 m) using a mobile phase consisting of acetonitrile/water (5 mM ammonium acetate in water, pH 6.2, 40:60, v/v) during a total run time of 4.0 min. The mass spectrometer was operated under the positive ionization mode. The multiple reaction monitoring with the transitions of m/z 248.1 174.2, 234.2 160.0 and 389.2 201.1 was used to quantify pethidine, norpethidine and cetirizine (internal standard), respectively. Linear calibration curves were obtained over concentration ranges of 8.248,440.00 ng/mL for pethidine, and 6.156,300.00 ng/mL for norpethidine. The intra- and interday precisions and accuracies were 15 (relative standard deviations, RSDs), and the accuracies were within the interval of 94103 for both. The lower limits of quantification in mouse plasma were 8.24 ng/mL for pethidine and 6.15 ng/mL for norpethidine. Subsequently, the validated method was successfully applied to a pharmacokinetics study of pethidine and its metabolite, norpethidine, in mice.