Neomangiferin, a natural C-glucosyl xanthone, has recently received a great deal of attention due to its multiple biological activities. In this study, a rapid and sensitive ultra-high performance liquid chromatography tandem mass spectrometry(UHPLC–MS/MS) method for the quantification of neomangiferin in rat plasma was developed. Using chloramphenicol as an internal standard(IS), plasma samples were subjected to a direct protein precipitation process using methanol(containing 0.05% formic acid). Quantification was performed by multiple reactions monitoring(MRM) method, with the transitions of the parent ions to the product ions of m/z 583.1-330.9 for NG and m/z 321.1-151.9 for IS. The assay was shown to be linear over the range of 0.2–400 ng/m L, with a lower limit of quantification of 0.2 ng/m L. Mean recovery of neomangiferin in plasma was in the range of 97.76%–101.94%. Relative standard deviations(RSDs) of intra-day and inter-day precision were both o 10%. The accuracy of the method ranged from94.20% to 108.72%. This method was successfully applied to pharmacokinetic study of neomangiferin after intravenous(2 mg/kg) and intragastric(10 mg/kg) administration for the first time. The oral absolute bioavailability of neomangiferin was estimated to be 0.53% 7 0.08% with an elimination half-life(t_(1/2)) value of 2.747 0.92 h, indicating its poor absorption and/or strong metabolism in vivo. Neomangiferin, a natural C-glucosyl xanthone, has recently received a great deal of attention due to its multiple biological activities. In this study, a rapid and sensitive ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS / MS) method for the quantification of neomangiferin in rat plasma was developed. Using chloramphenicol as an internal standard (IS), plasma samples were subjected to a direct protein precipitation process using methanol (containing 0.05% formic acid). Quantification was performed by multiple reactions monitoring (MRM) method, with the transitions of the parent ions to the product ions of m / z 583.1-330.9 for NG and m / z 321.1-151.9 for IS. The assay was shown to be linear over the range of 0.2-400 ng / ml , with a lower limit of quantification of 0.2 ng / m L. Mean recovery of neomangiferin in plasma was in the range of 97.76% -101.94%. Relative standard deviations (RSDs) of intra-day and inter-day precision were both o 10 %. The accuracy of th e method ranged from 94.20% to 108.72%. The method was successfully applied to pharmacokinetic study of neomangiferin after intravenous (2 mg / kg) and intragastric (10 mg / kg) administration for the first time. The oral absolute bioavailability of neomangiferin was estimated to be 0.53% 7 0.08% with an elimination half-life (t_ (1/2)) value of 2.747 0.92 h, indicating its poor absorption and / or strong metabolism in vivo.