The burden of depression and other mental disorders is on the rise globally, and successful treatment sometimes requires modifications of drugs and/or dose regimens. The development of novel analytical methods for the determination of antidepressant drugs in biological fluids is thus urgently required. Herein, a sensitive, robust, and rapid liquid chromatographic coupled tandem mass spectrometric method was developed and validated for the determination of citalopram (CIT) and sertraline (SER) in rat plasma after oral administration. The analytes of interest and internal standard (duloxetine (DUL)) were extracted from 100 μL of plasma with solid-phase extraction on an Oasis HLB cartridge followed by the separation with gradient elution with water containing 0.1% formic acid and acetonitrile on an Agilent Eclipse Plus ODS (4.6 × 100 mm, 3.5 μm) column at flow rate 0.2 mL min-1. The triple quadrupole mass spectrometry was applied via electrospray ionization source for detection. The fragmentation pattern of the protonated CIT, SER, and DUL was elucidated using multiple reaction monitoring of the transitions of m/z 325.2 to 109, 306.1 to 158.9, and 298.1 to 154.1 as [M + H]+ for CIT, SER, and DUL, respectively. The proposed method has been validated as per US-FDA bioanalytical guidelines in terms of linearity, accuracy, precision, matrix effects, stability, selectivity, and recovery. The method was linear over the concentration range of 1-2000 and 1-1000 ng mL-1 with the lower limit of detection of 0.12 and 0.19 ng mL-1 for CIT and SER, respectively. The interday and intraday precisions and accuracy expressed by the relative standard deviation and the relative standard error were both lower than 11.1% and 2.1%, respectively. The proposed method was successfully applied for the pharmacokinetics and drug monitoring studies of CIT and SER in rat plasma after a single oral dose of 120 mg kg-1 of CIT and SER. Coadministration of SER with CIT has affected the peak plasma concentrations, maximum plasma concentration time, area under the concentration-time curve, and oral clearance of CIT. Molecular modeling study showed that SER could competitively inhibit CYP2D6, the main enzyme involved in CIT metabolism. A possible drug-drug interaction in psychiatric patients undergoing chronic SER and CIT treatment is therefore worthy of more attention in an effort to avoid side effects and serotonin syndrome.