Adhatoda vasica (L.) Nees (Acanthaceae) is a well known plant drug in Ayurvedic medicine, used for the treatment of different types of diseases and disorders. The tissue cultures of this plant species were established on Murashige and Skoog culture medium by using various concentrations of growth hormones (0.5-5.0 mg/L α- napththaleneacetic acid + 0.5-3.5 mg/L 6-benzylaminopurine). The concentration of vasicine acetate was found higher (0.51 ÃÆÃÂ¯ÃâÃ¢â¬Å¡ÃâÃÂ± 0.519%) in six week old callus than in vivo (0.47 ÃÆÃÂ¯ÃâÃ¢â¬Å¡ÃâÃÂ± 0.556%) plant material (roots). The antioxidant activity was evaluated by using DPPHradical scavenging, reducing power and superoxide anion scavenging models. We found that 2-acetyl benzyl amine showed maximum antioxidant activity against various antioxidant models, including DPPH-radical scavenging (40.59 ± 0.774%), reducing power (461.55 ± 0.402%) and superoxide anion scavenging (52.54 ± 0.553%) than other isolated alkaloids at 10-30 μM concentrations. Our observed results suggest that the pyrroloquinazoline alkaloids of A. vasica demonstrated strong antioxidant activity, which could be used as potent drugs against different types of oxidative stresses.