<jats:title>Abstract</jats:title><jats:p>A sensitive and rapid liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of sertraline in human plasma. The analyte and internal standard (IS, diphenhydramine) were extracted with 3 mL of diethyl ether/dichloromethane (2:1, v/v) from 0.25 mL plasma, then separated on a Zorbax Eclipse XDB C<jats:sub>18</jats:sub> column using methanol/water/formic acid (75:25:0.1, v/v/v) as the mobile phase. The triple quadrupole mass spectrometry was applied via an atmospheric pressure chemical ionization (APCI) source for detection. The fragmentation pattern of the protonated sertraline was elucidated with the aid of product mass spectra of isotopologous peaks. Quantification was performed using selected reaction monitoring of the transitions of <jats:italic>m/z</jats:italic> 306 → 159 for sertraline and <jats:italic>m/z</jats:italic> 256 → 167 for the IS. The method was linear over the concentration range of 0.10–100 ng/mL. The intra‐day and inter‐day precisions, expressed by relative standard deviation, were both less than 6.7%. Assay accuracies were within ±6.9% as terms of relative error. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.10 ng/mL with a precision of 8.3% and an accuracy of 9.6%. The validated method has been successfully applied for the pharmacokinetic study and bioequivalence evaluation of sertraline in 18 healthy volunteers after a single oral administration of 50 mg sertraline hydrochloride tablets. Copyright © 2006 John Wiley & Sons, Ltd.