A novel rapid and highly sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) bioanalytical method was established for the analysis of flibanserin in human plasma. Flibanserin d4 was used as internal standard (IS). Flibanserin and the internal standard (IS) were extracted from the plasma using protein precipitation technique with acetonitrile. A Kinetex C 18 (2.6 μm, 2.1 × 50 mm) column was used for chromatographic separation and the mobile phase was a mixture of 20 mm ammonium acetate buffer (pH 4.5)-acetonitrile (50:50, v/v) with an isocratic elution mode and a flow rate of 0.3 mL/min. The analysis was performed on a Xefo TQD Waters mass spectrometer in multiple reaction monitoring mode with a positive electrospray ionization interface. The US Food and Drug Administration guidelines were followed during the bio-analytical methods validation regarding linearity, precision, accuracy, carryover, selectivity, dilution integrity and stability. The analysis run time was carried out within 2 min over a wide linear concentration range of 5-1000 ng mL -1 . Finally, the proposed method was successfully used in a pharmacokinetic study that measured flibanserin concentration in healthy, non-pregnant female volunteers after a single 100 mg oral dose of flibanserin., (© 2019 John Wiley & Sons, Ltd.)