Kinsenoside is a major bioactive constituent isolated from Anoectochilus formosanus and is investigated as an antihyperlipidemic candidate. In this study, a rapid, sensitive, and reliable bioanalytical method was developed for the determination of kinsenoside in rat plasma using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The plasma sample was pretreated with 1% acetic acid, followed by protein precipitation with acetonitrile:methanol (70:30). Chromatographic separation was performed on a HILIC silica column (2.1 mm × 100 mm, 3 μm). The mobile phases consisted of 0.1% acetic acid in distilled water (solvent A) and 0.1% acetic acid in acetonitrile (solvent B). A gradient program was used at a flow rate of 0.2 mL/min. For mass spectrometric detection, the multiple reaction monitoring mode was used; the MRM transitions were m/z 265.2 → m/z 102.9 for kinsenoside and m/z 163.3 → m/z 132.1 for the internal standard (IS) nicotine in the positive ionization mode. A calibration curve was constructed in the range of 2–500 ng/mL. The intra- and interday precision and accuracy were within 5%. The HILIC–MS/MS method was specific, accurate, and reproducible and was successfully applied in a pharmacokinetic study of kinsenoside in rats.