An ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of sitagliptin phosphate in rat plasma was established. The blank rat plasma sample added with sitagliptin phosphate and the internal standard (fluoxetine) standard solution were prepared. Methanol was added in the sample for the deproteinization. Then the sample was vortex-mixed and centrifuged. The clear supernatant was used for the analysis of UPLC-MS/MS. A Thermo Hypersil Gold C18 column (50 mm x 2.1 mm, 1.9 microm) was employed with a guard column of Phenomenex Security Guard C18 column (4 mm x 3.0 mm), and the column temperature was set at 35 degrees C. The gradient elution of acetonitrile and water (containing 0.05% (v/v) formic acid) as mobile phases was performed at a flow rate of 200 microL/min, and a rapid separation was completed in 5 min. The electrospray was operated in the positive ionization mode and the sitagliptin phosphate and fluoxetine were identified by selected reaction monitoring (SRM) mode, and the monitoring ions of them were m/z 408.0 --> 235.0 and m/z 310.0 --> 148.0, respectively, which were used to qualify and quantity the targets by the method of matrix-matched standard solution. The calibration curve showed good linearity within the concentrations of 1 to 1 000 microg/L (r = 0.999 1); the limit of detection was 0.2 microg/L. The mean recoveries were from 85% to 115% at the spiked levels of 5, 50 and 500 microg/L; the relative standard deviations (RSDs) of intra- and inter-day of variation were both less than 15%, which can meet the determination requirements of biological samples. Then the method was initially used for the determination of sitagliptin phosphate in SD rat plasma after the administration of a single intravenous injection dose of sitagliptin phosphate. The method is rapid, sensitive, convenient and reproducible in the determination of sitagliptin phosphate, and can be used for the pharmacokinetics research of sitagliptin phosphate in plasma.