Background: Calotropis gigantea (L.) W.T. Aiton, belonging to the family Apocynaceae, is a source of many bioactive cardiac glycosides. Accumulation of secondary metabolites in plants depends upon various external and internal factors, including the plant age or growth stage. Objectives: The main aim of this study was to identify the correct variant and growth stage of C. gigantea for the maximum yield of targeted metabolites. Materials and Methods: In the present experiment, three different tissues, that is, leaf, stem, and root, of 3–12-month-old white flower variant (WFV) and purple flower variant (PFV) of C. gigantea were investigated to find out the growth stage-specific accumulation pattern of five cardiac glycosides (CGs) and their three genins. Results: Highest concentrations of calotropin, uscharin, and uscharidin were found in the stem of five-month-old WFV. Frugoside and uzarigenin were at peak levels in the stem of a nine-month-old member of the same variant. Calotropagenin and coroglaucigenin were at maximum levels in the root and leaf of 8- and 10-month-old members, respectively, of this variant too. The only CG accumulated at maximum level in the root of 11-month-old PFV was asclepin and its quantity was comparable to 8-month-old WFV root. Conclusion: It was observed that the WFV was superior to PFV, considering the accumulation of most of the CGs. Therefore, it can be concluded that the specific tissues of 5–10-month-old members of WFV are optimum for an economic yield of respective CGs.