Shoot cultures of medicinally acclaimed plant Boerhaavia diffusa were initiated in vitro and the influence of different strengths of Murashige and Skoog (MS) medium, plant growth regulators, carbon/nitrogen/phosphate (CNP ratio) and varying concentrations of carbon sources on biomass regeneration and boeravinone B production were analyzed. Experiments were performed to examine the effect of media manipulation strategies through HPTLC analysis on a useful secondary metabolite- boeravinone B in the plant species. MS full strength medium fortified with zeatin (Zn) (1 mg/L) and naphthalene acetic acid (NAA) (0.5 mg/L) indicated complete success with an average of 13.0 ± 0.33 shoots per explant and 10 -14 roots per explant were obtained on medium supplemented with NAA (0.5 mg/L). Well rooted plantlets were hardened and acclimatized with 95 % survival rate under green house conditions. CNP ratio of 1:1:1 (control) emerged as the best combination for biomass production (4.58 DW) in this plant species. A CNP ratio of 2:1:1 exhibited higher boeravinone B content of 16.78 %, wherein the concentration of carbon source was increased two folds. Likewise, by increasing the table sugar concentration upto 5 %, highest boeravinone B content (19.15 %) was recorded, which is 3.9 times more than that in the field grown plants. Since, sucrose (5.58 DW) and table sugar (5.63 DW) at 3 % concentration showed similar results for biomass production so sucrose in the medium could be replaced by a cheaper carbon source (table sugar) to make process cost-effective. This protocol can be used for maximizing biomass and metabolite content for its commercial exploitation and applications in herbal drug based industries.