A sensitive, selective, and rapid high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the quantification of rohitukine in rat plasma. HPLC was performed using a Symmetry-Shield C18 (5 μ, 4.6 × 150 mm) column, and isocratic elution with ammonium acetate buffer (pH4; 10 mM):methanol (08:92, v/v) at a flow rate of 0.6 mL/min. Sample clean-up involved solid phase extraction (SPE) of analyte and internal standard (phenacetin) from 100 μL plasma. The parent→product ion transitions (MRM) for analyte and IS were 306.1→245.1 m/z and 180.1→138.1 m/z respectively, and were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The method was validated across the dynamic concentration range of 5-500 ng/mL for rohitukine, with a fast run time of 4.5 min. The analytical method measured concentrations of rohitukine with accuracy (% bias) of <±10% and precision (% RSD) of <±12%. Rohitukine was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days of storage in a freezer at -70±10°C. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic and in vitro metabolism studies in Sprague-Dawley rat. This method will therefore be highly useful for future preclinical and clinical pharmacokinetic studies of rohitukine.