A sensitive and credible high performance liquid chromatography hyphenated to mass spectrometry (HPLC–MS/MS) was established to quantify the concentration of gracillin in rat plasma. The plasma samples were subjected to a direct protein precipitation process with acetonitrile as a precipitant in a single-step. Ginsenoside Rb1 was selected as an internal standard (IS). The chromatographic separation of analyte and IS were carried out on an Inersil ODS-3 C18 column (250 × 4.6 mm, 5 μm) with a binary solvent system containing acetonitrile and 0.1% formic acid in water at a flow rate of 1 mL min−1 under a gradient elution mode. Mass spectrometric detection was performed on a triple quadrupole tandem mass spectrometer by the multiple reaction monitoring (MRM) mode to examine the precursor-to-daughter ion transitions of 1110.3 → 948.2 for IS and 886.1 → 739.9 for gracillin, respectively, in a positive electrospray ionization mode. The calibration curve showed a promising linearity over a concentration range of 0.065–800 ng mL−1 with a better regression coefficient of r2 = 0.9960. The intra- and inter-day precisions (as relative standard deviation) of the assay at three quality control levels were all less than 3.48%, while the intra- and inter-day accuracies (as relative error) ranged from −8.43% to 9.74%, whose data were within the acceptable limits. The mean extraction recoveries of analyte from rat plasma were all more than 74.11%, and no notable matrix effect was observed. Stability experiments revealed that gracillin remained stable throughout the analytical procedure under various stored conditions. The above validated method was successfully used to investigate the pharmacokinetic behaviors of gracillin orally administrated to rats at three proportion doses. The pharmacokinetic analysis would pave the way for understanding the pharmacological actions and provide a meaningful foundation for further development and application in preclinical and clinical use of gracillin in the near future.