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A method for the analysis of mangiferin and neomangiferin was developed.The method produced a linear range for each drug in rat plasma.The method offered higher sensitivity and shorter run time of 3.0 min.In this study, a sensitive and rapid ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method was developed to determine mangiferin and neomangiferin in rat plasma simultaneously. Chromatographic separation was carried out on an Acquity UPLC BEH C18 column and mass spectrometric analysis was performed using a Xevo TQD triple quadruple mass spectrometer coupled with an electrospray ionization (ESI) source. The MRM transitions of m/z 423.2 → 303.1 and m/z 585.0 → 273.1 were used to quantify for mangiferin and neomangiferin, respectively. The linearity of this method was found to be within the concentration range of 5–2000 ng/mL for mangiferin, and 2–1000 ng/mL for neomangiferin in rat plasma, respectively. Only 3.0 min was needed for an analytical run. This assay was used to support a preclinical study to investigate the pharmacokinetics of mangiferin and neomangiferin in rats.