Lupeol, a phytosterol and triterpene, possesses numerous medicinal properties against cancer, inflammation, arthritis, diabetes, heart diseases, etc. A novel, sensitive, specific and reproducible method for quantification of Lupeol in rat plasma using liquid chromatography combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-MS/MS) was developed and validated as per regulatory guidelines. Sample preparation was simple and fast which consisted of one-step protein precipitation using acetonitrile. Testosterone was used as an internal standard. HyPurity Advance column was used to develop the chromatography method using 0.1% formic acid in water and acetonitrile as mobile phases by gradient elution. APCI positive ion mode was used for mass spectrometric detection. Multiple reaction monitoring (MRM) transitions of m/z 409.5 [M + H - H2O]+→137.3 for Lupeol and m/z 289.1 [M + H]+→97.1 for Testosterone were used for quantification. The method was validated over a linear concentration range of 5-5000 ng/mL with a correlation coefficient (r2) of ≥ 0.99. This method showed acceptable accuracy (89.52-97.10%), precision (%CV ≤ 10.75%) and recovery with a negligible matrix effect. Lupeol was found to be stable in the stock solution, autosampler condition and also in plasma for four freeze-thaw cycles, 6 h at ambient temperature and 30 days at -20°C. This method was successfully applied to measurement of Lupeol in plasma samples from pharmacokinetic study in rats and can be easily extended to human pharmacokinetic studies.