The response of adventitious root induction from cotyledonary nodes of soybean in different lighting conditions, and different concentrations of indole-3-butyric acid (IBA) were studied. Among the different lighting condition of explants placed in MS medium supplemented with IBA (1 mg/l), 16-hour photoperiod showed effective adventitious root induction when compared to explants placed in complete darkness. The total phenolic (166.6 mg/g) and flavonoid content (677mg/g) was higher in adventitious roots derived from cotyledonary node explant placed in MS medium supplemented with IBA 1mg/ml in 16 h photoperiod when compared to control roots (phenolic content (33.70 mg/g) flavonoid content (48.0 mg/g). The HPLC analysis confirmed that the quercetin content was improved in adventitious roots (293.3 mg g−1) from cotyledonary node explant of IBA 1mg/l in 16-hour photoperiod compared to control roots (31.9 mg g−1) in MS medium without any hormones. On an average, 9.1-fold increase in quercetin was achieved in adventitious roots of cotyledonary node explant grown in medium containing IBA (1mg/l) in 16h photoperiod compared to control. The antioxidant activity of adventitious roots improved as the volume of extracts used for assay was increased from 20 to 100 μl. In addition, the antioxidant potential of adventitious roots was significantly higher when compared to control. In DPPH assay, adventitious roots extracts (20, 40, 60, 80 and 100 μl) showed higher potency of inhibition when compared to the control. Hence, the adventitious root extracts were able to scavenge DPPH radicals in a concentration dependent manner.