Turkish galls (TG) is a traditional Uygur medicine typically used in clinics for dental disease and chronic ulcerative colitis. In this study, a novel liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of gallic acid, methyl gallate, and 1,3,6-tri-O-galloyl-β-d-glucose in rat plasma, which are the major bioactive compounds of TG. After a feasible protein precipitation using acetonitrile for sample preparation, chromatographic separation was performed with a BDS Hypersil C18 column (2.1 × 100 mm, 5 μm) at 30°C, and water containing 10 mmol of ammonium acetate and acetonitrile was used as the mobile phase with a flow rate of 0.3 mL/min. The MS detector was operated in the selective reaction monitoring with negative-ionization mode. The results of the method validation, including selectivity, linearity, accuracy, precision, extraction recovery, matrix effect, and stability of the compounds in the biosamples, were all within the current acceptance criteria. The established method was successfully applied to the pharmacokinetics study of three analytes in rats after an oral administration of TG extract and laid the foundation for studying the active components and mechanism of TG in vivo.© 2020 John Wiley & Sons, Ltd.