A sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of quetiapine in rat plasma. Following liquid-liquid extraction, the analyte was separated using a gradient mobile phase on a reverse-phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 384 to m/z 221 for quetiapine and m/z 327 to m/z 270 for the internal standard. The assay exhibited a linear dynamic range of 0.25-500 ng/mL for quetiapine in rat plasma. The lower limit of quantification was 0.25 ng/mL with a relative standard deviation of less than 7%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated method was successfully used to analyze rat plasma samples for application in pre-clinical pharmacokinetic studies. This method in rodent plasma could be adapted for quetiapine assay in human plasma.