A simple and sensitive method for separation and determination of harmol, harmalol, harmine and harmaline has been developed and validated. Harmol, harmalol, harmine and harmaline were separated using a Metasil ODS column by isocratic elution with flow rate 1.5 ml/min. The mobile phase composition was Isopropyl alcohol Á/Acetonitrile Á/ Water Á/Formic acid (100:100:300:0.3) (v/v/v/v) and pH adjusted 8.6 with triethylamine. Spectrophotometric detection was carried out at 330 nm. The linear range of detection for harmol, harmalol, harmine and harmaline were between 9.375 Á/250, 30.750 Á/246, 31.250 Á/500 and 31.000 Á/248 mg/ml, respectively. The method described was suitable for the determination of harmol, harmalol, harmine and harmaline in the seeds of Peganum harmala L.