AbstractDiosmetin, 3′,5,7‐trihydroxy‐4′‐methoxyflavone, is the aglycone of the flavonoid glycoside diosmin that occurs naturally in foods of plant origin. Diosmin exhibits antioxidant and anti‐inflammatory activities, improves venous tone and it is used for the treatment of chronic venous insufficiency. Diosmin is hydrolyzed by enzymes of intestinal microflora before absorption of its aglycone diosmetin. A specific, sensitive, precise, accurate and robust HPLC assay for the determination of diosmetin in human plasma was developed and validated. Diosmetin and the internal standard 7‐ethoxycoumarin were isolated from plasma by liquid–liquid extraction and separated on a C8 reversed‐phase column with methanol–water–acetic acid (55:43:2, v/v/v) as the mobile phase at 43°C. Peaks were monitored at 344 nm. The method was linear in the 10–300 ng/mL concentration range (r > 0.999). Recovery for diosmetin and internal standard was greater than 89.7 and 86.8%, respectively. Intra‐day and inter‐day precision for diosmetin ranged from 1.6 to 4.6 and from 2.2 to 5.3%, respectively, and accuracy was better than 97.9%. Copyright © 2004 John Wiley & Sons, Ltd.