An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method employing electrospray ionization (ESI) has been developed for the determination of mangiferin in rat plasma using diphenhydramine as the internal standard (IS). Liquid-liquid extraction (LLE) was used for sample preparation and the analysis was achieved with gradient elution on C(18) reversed phase column. The method was validated over the concentration range 0.02-5.0 microg/mL for oral administration and 0.4-100 microg/mL for intravenous administration. The intra-day and inter-day precision of mangiferin expressed as RSD<15% and the accuracy (RE) did not exceed 15%. This validated method is a novel technique for sample preparation and quantitation, which was successfully applied to estimate the bioavailability of mangiferin.