Onions (Allium cepa L.) are the rich source of flavonoids, consisting mainly quercetin-3,4´-O-diglucoside (QDG) and quercetin-4´-O-monoglucoside (QMG), as major flavonoids. In the present study, we report the in vitro protective effect of different layers of onion extract on biomarkers of oxidative stress and on free radical induced oxidative hemolysis in erythrocytes. Results show a significant protection of oxidative stress by onion extract on erythrocyte subjected to tert-butyl hydroperoxide (t-BHP) treatment, as evidenced by the decrease in malondialdehyde (MDA) and increase in reduced glutathione (GSH) content. Onion extracts also showed significant protection of the erythrocyte from oxidative hemolysis. Quercetin (at micromolar concentration) showed significant antioxidant effect in protecting erythrocytes from t-BHP induced oxidative changes. The results are more pronounced for outer layers as compared to inner layers, suggesting that the outermost living layers had higher antioxidant activities compared to innermost layers.