Strobilanthes kunthiana (Neelakurinji) is an endemic and underexploited plant belongs to the Family Acanthaceae. The aim of our study was to evaluate phytochemical analysis, GC-MS analysis and antioxidant activity of in vitro callus extract of Strobilanthes kunthiana. In this present study the phytochemical analysis of various extract of S. kunthiana in vitro callus were studied. Phytochemical analysis confirmed the presence of alkaloids, flavonoids, glycosides, saponins, tannins, steroids, terpenoids and phenols in the methanolic extract comparing to the other extracts. In this study GC-MS analysis revealed the presence of 10 bioactive phytochemical compounds were identified in the methanolic extract. The prevailing compounds were 9,12-octadecadienoic acid (Z,Z) (50.32%), hexadecanoic acid, methyl ester (20.69%), 9-octadecenoic acid (Z)-,methyl ester (10.45%), heptadecanoic acid, 16-methyl-, methyl ester (5.78%), 2,6-bis(1,1-dimethylethyl)-4-methyl phenol (4.70%). 3-methyl-2-ketobutyric acid tbdms (2.79%), 2,2,3,4-tetramethyl-5-hexen-3-ol (2.41%), N-(tertbutoxycarbonyl)-2-(4-methoxyphenyl) allylamine (1.06%), cyclotrisiloxane, hexamethyl (0.94%), benzenesulfonamide (0.87%). The antioxidant property was evaluated for methanol and ethanolic extract by DPPH method. The higher percentage of inhibition (79.23 ± 0.37) was observed in 250 µg/mL of ethanol extract followed by (90.35 ± 0.54) methanolic extract against the standard ascorbic acid (91.25 ± 0.33). The results show that the methanolic extract possesses more antioxidant activity than ethanol. The plant S. kunthiana may be exploited as a source of natural antioxidant and as herbal alternatives for various disorders.