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Different Podophyllum hexandrum explants have been used to establish callus cultures. Different levels of important medicinal substances with high pharmacological value such as those with total phenolics, total flavonoids, total antioxidants and anti-microbial activities are found in Podophyllum hexandrum. Although the presence of podophyllotoxin had already been reported but the micropropagation technique for plant growth and plant regeneration is not yet optimized. Few reports available which are non-reproducible and podophyllotoxin production under in vitro condition is not showing demarcated significance. So the sustained growth and regeneration of callus tissue were successfully accomplished using a fully defined medium composed of Murashige and Skoog salts and supplemented with GA₃ (0.5mg/L), IAA (2mg/L), TDZ (1mg/L), 2,4-D (1mg/L) and BAP (1mg/L). In the current study, a variety of stimulating chemicals (elicitors) were used to increase the synthesis of podophyllotoxin as well as biomass growth. Flavonoids, terpenes, saponins, and glycoside chemicals were detected in the phytochemical screening of a variety of methanolic extracts. The methanolic extract of red+green light treated callus produced the best results in terms of total phenol, total glycoside, anti-oxidant activity (DPPH assay), and Well diffusion and MIC techniques.