Natural products have been considered for many years ago to become a rich source of novel chemical structures. The present study was aimed to isolate a pure compound with significant antioxidant activity from Helianthus annuus leaves. The plant material was extracted successively by Soxhlet apparatus by using petroleum ether, chloroform, and methanol. These extracts were tested in vitro by using 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The antioxidant activity of methanol extract was (55± 0.05 RSA %) at 500 μg/ ml. Column chromatography was used to separate the methanol extract into eight fractions. The fractions were again tested by using DPPH radical scavenging assay, fraction seven (F7) has shown the highest ability to reduce DPPH with percentage 65% at 500 μg/ ml, it was even higher than the radical scavenging activity of the whole crude methanol extract. The IC50 of fraction seven has shown 329 μg/ ml. TLC technique was applied to confirm the purity of fraction seven. Cyanidin reaction and AlCl3 colorimetric assay method were applied to confirm that the isolated compound was a flavonoid most probably a flavone type. The isolated flavonoid compound is now under the process of determination of its chemical structure.