Alogliptin, as dipeptidyl peptidase (DPP-4) inhibitor, is used for Type 2 diabetes mellitus in many counties worldwide. In this work, a sensitive and selective ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determination of alogliptin in rat plasma was developed and validated. After addition of diazepam as an internal standard (IS), protein precipitation by acetonitrile-methanol (9: 1, v/v) was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 x 100 mm, 1.7 mu m) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reactions monitoring (MRM) mode was used for quantification using target fragment ions m/z 340.2 -> 116.0 for alogliptin, and m/z 285.1 -> 193.1 for IS. Calibration plots were linear throughout the range 2-2000 ng/mL for alogliptin in rat plasma. Mean recoveries of alogliptin in rat plasma ranged from 81.5% to 91.4%, matrix effect of alogliptin in rat plasma ranged from 105.9 to 110.5%. RSD of intra-day and inter-day precision were both <10%. The accuracy of the method was between 95.2% and 110.3%. The method was successfully applied to pharmacokinetic study of alogliptin after either oral or intravenous administration. The absolute bioavailability of alogliptin was reported as high as 30.9%.