A sensitive gas chromatography–time-of-flight mass spectrometry (GC–ToFMS) method has been developed and validated for the determination of buagafuran, a new anxiolytic drug derived from α-agarofuran. Buagafuran and internal standard (buagafuran-d4) were isolated from plasma by liquid–liquid extraction. The separation was achieved on HP-1 capillary column (25 m × 0.2 mm × 0.11 μm). Buagafuran (m/z 262.22) and buagafuran-d4 (m/z 266.25) were eluted at 7.6 min and no endogenous materials interfered with the measurement. The calibration curves of buagafuran were linear over the range of 2.5–160 ng/ml in dog plasma. The lower limit of quantification (LLoQ) was 2.5 ng/ml in plasma. The within-day and between-day precisions were less than 15%. The method was used to determine the plasma concentration–time profile of buagafuran after oral doses of 8, 16, 32 mg/kg in dogs.