Objective: An efficient in vitro plant regeneration protocol for Canscora decussata(Gentianaceae), (C. decussata) a threatened medicinal herb used in Ayurvedic system of medicine was developed. Seed germination was achieved on MS growth regulator free medium. Methods: The nodal explants were excised from the in vitro raised seedlings and inoculated on MS medium supplemented with various plant growth regulators such as BAP, KIN, TDZ and Zeatin individually and in combinations with or without GA3. BAP (2 mg/L) was proved to be effective for multiple shoot induction (30.20±6.53) among the cytokinin tested individually. Addition of NAA (1 mg/L) to cytokinin containing medium resulted in callus, with KIN (3 mg/L) produced highest percentage of callus (82%) per explant. Results: Among the various combination of cytokinin tested, BAP (0.5 mg/L) in combination with KIN (2 mg/L) induced highest number of multiple shoots (72.10±1.05 shoot per explant). Addition of 1 mg/L of GA3 to the above medium induced highest number of shoots (100.80±3.20) with an average shoot length of (6.98±0.66 cm). Rooting was optimized in half-strength MS medium supplemented with IBA at 1.0 mg/l. The plantlets were successfully transferred to hardening medium containing vermiculite with 83% survival rate. Among the antioxidant activity of methanol extract of wild-grown plants and in vitro regenerants tested, half-MS medium supplemented with NAA (0.5 mg/L) derived callus has promising activity for total phenolics, DPPH, ABTS, FRAP and phosphomolymbdenum assays. Total flavonoid content was found to be high in callus derived from MS medium supplemented with KIN (2 mg/L) in combination with NAA (1 mg/L). Conclusion: Our present study suggest that in vitro derived callus of C. decussata represent a promising altative source to meet the pharmaceutical demands for commercial formulations and the protocol could effectively be applied for the conservation of C. decussata.