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A simple, accurate and selective HPLC method was developed and validated for determination of quercetin and kaempferol, which are the main flavonols in broccoli. The separation was achieved on a reversed-phase C18 column using a mobile phase composed of methanol/water (60/40) and phosphoric acid 0.2% at a flow rate of 1.0 ml min-1. The detection was carried out on a DAD detector at 370 nm. This method was validated according to the requirements for new methods, which include selectivity, linearity, precision, accuracy, limit of detection and limit of quantitation. The current method demonstrates good linearity, with R2 > 0.99. The recovery is within 98.07-102.15% and 97.92-101.83% for quercetin and kaempferol, respectively. The method is selective, in that quercetin and kaempferol are well separated from other compounds of broccoli with good resolution. The low limit of detection and limit of quantitation of quercetin and kaempferol enable the detection and quantitation of these flavonoids in broccoli at low con–centrations

Validation of the Analytical Method for the Determination of Flavonoids in Broccoli