Dapoxetine is an oral medication used for treatment of premature ejaculation (PE) in men aged (18–64 years). In this study, we present a validated, precise and sensitive method for determination of dapoxetine in human plasma by liquid chromatography/ electrospray ionization-tandem mass spectrometry. Dapoxetine and the internal standard (Dapoxetine- d6) were extracted from plasma via liquid–liquid extraction (LLE). The LC separation was performed utilizing ACE C8 (4.6 X50) mm, 5 µm column. The mobile phase was composed of acetonitrile and buffer (0.01 M Ammonium acetate +0.02% Formic acid solution) (85:15, v/v). The method was linear within the concentration range of 5.0–600 ng/mL for Dapoxetine in human plasma. Short analytical run was achieved with 1.6 min run time. Intra-day and inter-day accuracy was between 97 and 106% with precision (CV, %) of ≤ 5% achieved across all the quality control samples. Dapoxetine was stable in several conditions with recovery rates > 90%. This method was utilized successfully in clinical pharmacokinetic study following oral administration of 60 mg Dapoxetine tablets in 36 healthy male subjects. The result for all 90% confidence intervals were within the preset ranges.The method proved to be highly reproducible and sensitive and thus can be employed in bioequivalence studies and large scale sample analysis of Dapoxetine.