A procedure for determining brimonidine [5-bromo-6-(2-imidazolidinylideneamino) quinoxaline] in biological samples using a reversed-phase isocratic HPLC method is described. The application in blood serum and eye aqueous humor of patients treated with the Alphagan ophthalmic solution was carried out by enrichment of samples in brimonidine with solid-phase liquid extraction. Brimonidine reached maximum levels in aqueous humor and serum within 2-2.5 h, whereafter a declining pattern was obtained. An approximate 50% level of brimonidine was identified in serum at 12 h after ocular administration, whereas in aqueous humor this percentage was determined after a period of 4-5 h.