Ophiorrhiza mungos (O. mungos), a significant medicinal plant belonging to the Rubiaceae family, is native to the Indian subcontinent and predominantly found in countries like India, Nepal, Bhutan, Bangladesh, and Sri Lanka. Recently, it has gained recognition as a promising alternative source of anticancer compounds. The presence of camptothecin (CPT), a modified monoterpene indole alkaloid, in notable quantities has sparked interest in researching O. mungos. This present study is focused on enhancing the synthesis of CPT through in vitro shoot growth of O. mungos using a half-strength Murashige and Skoog growth medium supplemented with specific precursors. Among the precursors tested, L-tryptophan and geraniol exhibited the most significant increase in CPT levels, with a 33 % enhancement compared to the untreated shoots. The researchers employed response surface methodology to explore the combined effect of these precursors on CPT production. In the study, in vitro plantlets were treated with L-tryptophan and geraniol at a concentration of 1.5 mM each, resulting in the highest CPT levels ranging from 1658.52 to 1747.63 µg/g. The validity of the experimental approach was confirmed by the close agreement between the experimental value and the predicted value of 1702.00 µg/g, demonstrating a 99.3 % validity of the model. This study underscores the significance of utilizing a combination of precursors to enhance the production of secondary metabolites, specifically CPT, in O. mungos shoot cultures. Furthermore, it identifies L-tryptophan and geraniol, along with their respective doses, as effective precursors for improved CPT synthesis. Notably, this research employed response surface methodology to investigate the potential of L-tryptophan and geraniol as precursors for CPT production in the in vitro shoot growth of O. mungos, marking a significant contribution in this field of study.Top of Form Display omitted •Precursor feeding of Ophiorrhiza mungos in vitro shoots to enhance camptothecin production.•The potential of L-tryptophan and geraniol as precursors for camptothecin production.•L-tryptophan and geraniol at a concentration of 1.5 mM each, for increased camptothecin levels in Ophiorrhiza mungos in vitro shoots.