A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile-methanol-water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl-sn-glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025-0.4 mg/ml sphingomyelin (R2>0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.