2-[[(1,1-dimethylethyl)oxidoimino]methyl]-3,5,6-trimethylpyrazine (TBN), a novel nitrone derivative of tetramethylpyrazine (TMP), was found to be a potent candidate compound for ischemic stroke treatment. It is currently in preclinical development as a stroke therapeutic. To study its pharmacokinetic characteristics, a simple and rapid HPLC-UV method was developed and validated to quantitatively determine TBN concentration in rat plasma. A Purospher C(18) column (150 × 4.6 mm, 5 μm) was used for analysis with a mobile phase containing methanol-potassium dihydrogen phosphate buffer solution (50 mM, pH 3.0) (45:55, v/v) and UV detection at 295 nm. The pharmacokinetic study was conducted in Sprague-Dawley rats by intravenous (i.v. 40, 80, and 160 mg/kg) and intragastric (i.g. 80 mg/kg) administration. The concentration-time profiles of TBN in plasma fitted a two-compartment model for both administration routes. The elimination half-life (T (1/2) (β)) of i.v. administration ranged from 134 to 225 min for low, middle and high dosage, and the area under the concentration-time curve from zero to infinity (AUC((0-∞))) ranged from 7,954 to 49,804 μg min/mL. Compared with the parent compound TMP, TBN showed a longer T (1/2) (β) (TBN 134.52 min, TMP 91.85 min) and a higher AUC((0-∞)) (TBN 22,687.84 μg min/mL, TMP 7,287.98 μg min/mL) after the same dosage of intravenous administration (80 mg/kg). The intragastric administration of TBN had a peak time of 21.65 min, C (max) of 41.71 μg/mL, and k (a) of 0.19 min(-1). And the absolute bioavailability was 36.02%.