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-Acetonitrile-Water-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 9.375-250, 30.750-246, 31.250-500 and 31.000-248 mug/ml, respectively. The method described was suitable for the determination of harmol, harmalol, harmine and harmaline in the seeds of Peganum harmala L. (C) 2002 Elsevier Science B.V. All rights reserved.