Background: Calotropis gigantea (L.) Dryand, belonging to the family Apocynaceae is a source of many bioactive cardenolides. However, inadequate accumulation of these cardenolides within this plant restricts their economic isolation. Due to multiple chiral centers, these metabolites could not be chemically synthesized so far. Objective: The main objective of this study was to develop protocols for enriched production of targeted bioactive cardenolides within in vitro grown plant system. Materials and Methods: In our study, we established in vitro plantlets from the seeds of naturally grown plants. Various biotic and abiotic elicitors, as well as precursors with different concentrations and incubation period, were used for the same. Results: It was observed that the in vitro grown control plantlets could biosynthesize a higher quantity of seven bioactive cardenolides than the naturally grown plants, whereas only coroglaucigenin was in less quantity. All the eight cardenolides could further successfully be enriched through our experiments. Uscharidin could be enriched significantly to a maximum level (~17-fold) followed by calotropagenin (~11-fold), uzarigenin (~8.5-folds), calotropin (4.5-fold), frugoside (~4-fold), uscharin (3.8-fold), asclepin (~2-fold), and coroglaucigenin (~1.5-fold) when they were compared to their maximum accumulation in naturally grown plants. For effective quantitative calculation of natural abundance of cardenolides within naturally grown plant, their seasonal variations were carried out using ultra high-performance liquid chromatography-mass spectrometry. Conclusion: From the above results, it can be concluded that the in vitro grown plantlets are the better choice than the naturally grown plants for enriched production of cardenolides. Elicitors were found more effective than precursors for the same.