Hemidesmus indicus (L.) R.Br. (Asclepiadaceae), commonly known as Anantamul, is an endangered, high value medicinal climbing shrub renowned for its therapeutic bioactives, including 2-hydroxy-4-methoxybenzaldehyde. Destructive wild harvesting and the inherent limitations of conventional seed propagation present severe challenges to its conservation, requiring the development of robust in vitro regeneration and biochemical evaluation protocols. The primary objective of this study was to establish an optimized, highly reproducible in vitro micropropagation protocol for H. indicus using varying explants, alongside a comparative qualitative phytochemical screening of in vitro regenerated plantlets versus naturally grown plants. Nodal, leaf and shoot apices segments were aseptically cultured on Murashige and Skoog (MS) media supplemented with varied combinations of auxins (2,4-D, NAA, IBA) and cytokinins (BAP, Kn). Qualitative phytochemical screening was performed on methanolic extracts of both in vivo and in vitro plantlets using standard colorimetric assays for alkaloids and ten other secondary metabolites. Optimal callus induction was achieved in nodal (96%) and leaf (95%) segments utilizing MS medium fortified with 1.0 mg/l 2,4-D or a combination of 1.5 mg/l BAP and 1.0 mg/l NAA. Direct multiple shoot regeneration from nodal segments peaked at 95% (yielding 5.44 ± 0.35 shoots/explant) on MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l Kn. Shoot apices produced a maximum of 3.54 ± 0.29 shoots/explant on MS medium with 2.0 mg/l BAP and 1.0 mg/l Kn. The greatest shoot elongation (5.22 ± 0.15 cm) was achieved using 1.0 mg/l BAP and 0.5 mg/l NAA. Maximum rooting (85%; 5.52 ± 0.04 roots/shoot) was recorded on full strength MS medium supplemented with 1.5 mg/l IBA. Following hardening, ex vitro acclimatization achieved an 85% survival rate. Phytochemical profiling confirmed that in vitro regenerated clones accumulated substantially higher concentrations of coumarin, quinine and phlobatannin compared to naturally grown specimens, while consistently maintaining high levels of steroids, flavonoids and terpenoids. The optimized propagation protocol establishes a rapid, reliable and sustainable pathway for the mass multiplication of H. indicus. The enhanced accumulation of critical pharmacological metabolites in in vitro tissues confirms that tissue culture represents a superior strategy for both ecological conservation and commercial pharmaceutical exploitation.