The aim of this study was to develop and fully validate a sensitive and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantification of pristinamycin ⅠA (PⅠA) and pristinamycin ⅡA (PⅡA) in plasma of beagle dogs after oral administration of pristinamycin tablets. PⅠA, PⅡA and quinupristin (internal standard, IS) were separated on an Agilent Eclipse Plus C18 column (2.1 mm × 100 mm, 3.5 μm particle size) by using gradient elution consisting of methanol and water (0.1 % formic acid) at a flow rate of 0.4 mL/min in 4.0 min. Multiple reaction monitoring (MRM) mode was performed to quantify data under monitoring precursor-product ion transitions of m/z 867.6→134.1, 548.4→287.1 and 1022.7→133.9 for PⅠA, PⅡA and IS at positive ion mode, respectively. The method was developed at linearity ranging from 1.0 to 1000 ng/mL for all analytes.The accuracy of PⅠA and PⅡA was observed to range between -10.6 % and 7.1 %, while the precision was found to be within 8.9 %. No significant matrix effect was observed. PⅠA and PⅡA demonstrated stability during sample storage, preparation and analytic procedures. Furthermore, this method was successfully applied in the investigation of the pharmacokinetic profile of PⅠA and PⅡA in beagle dogs after oral administration of pristinamycin tablets (75 mg for PⅠA and 175 mg for PⅡA). The biological half-life (t1/2) was determined to be 1.75 ± 0.07 h and 1.44 ± 0.31 h for PⅠA and PⅡA, respectively. The areas under curves (AUC0-t) of PⅠA and PⅡA were 80.7 ± 24.6 and 230 ± 94.8 μg/L·h, respectively.