A highly sensitive ultra-high pressure liquid chromatography (UHPLC-MS/MS) method for estimation of paclitaxel in rat plasma has been validated. The chromatographic separation was achieved using isocratic mobile phase, consisting of acetonitrile–2mM ammonium acetate (70:30 v/v; 0.25 mL min-1). Docetaxel was used as an internal standard and tert-butyl methyl ether (TBME) as organic solvent for the liquid-liquid extraction. The mass spectrometer was operated in synapt mass spectrometry mode via positive electrospray ionization using the transitions m/z 854.4 → 286.1for paclitaxel, and m/z 808.3→ 527.2 as a parent ion of docetaxel (IS). The mean extraction-efficiency of QC samples was 90%. The lower limit of quantification (LLOQ) was 1.0 ng/mL. This method covered a linearity range from 1- 5000 ng/ml, with the total run time of 3.0 min. The applicability of UHPLC-MS/MS method was demonstrated and successfully applied for pharmacokinetic profiling of paclitaxel in rat plasma after oral administration.