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Objective: To simultaneously investigate the pharmacokinetics of gentiopicroside, sweroside, and swertiamarin, which are constituents of Gentianella acuta, by developing and validating a simple, sensitive, and fast ultra?high?performance liquid chromatography–tandem mass spectrometry method. Materials and Methods: Blood samples were collected from the forward limb veins of six beagle dogs following oral gavage with G. acuta, the whole plant extract (39.90 mg/kg). Plasma samples were processed using liquid–liquid extraction. The analytes and paeoniflorin (internal standard [IS]) were separated using an Acquity? UPLC ethylene bridged hybrid amide column (2.1 mm × 100 mm, 1.7 μm) with isocratic elution using a mobile phase consisting of acetonitrile and 0.1％ formic acid in water (80:20, v/v) at a flow rate of 0.4 mL/min. Quantification was performed using multiple reaction monitoring of the fragmentation transitions at m/z 401.1→179.0, 403.1→195.0, 419.1→179.0, and 525.2→449.1 for gentiopicroside, sweroside, swertiamarin, and the IS, respectively. Results: The linearity of the analytical response was good and the calibration curves were linear over concentration ranges of 1.20–192.0, 0.40–159.0, and 0.20–209.3 ng/mL for gentiopicroside, sweroside, and swertiamarin, respectively. The extraction recovery was in the range of 84.72％–91.34％, 84.58％–93.43％, and 82.75％–91.37％ for gentiopicroside, sweroside, and swertiamarin, respectively. Conclusions: The method was successfully used to evaluate the pharmacokinetic parameters of gentiopicroside, sweroside, and swertiamarin in beagle dogs.