Background: Dendrobium thyrsiflorum is highly vulnerable to extinction in its natural habitats due to overexploitation for the medicinal and horticultural purposes. The poor seed germination in a natural condition and slow plant production through vegetative approach make in vitro micropropagation techniques an ideal choice for effective plant propagation. There are very limited reports on the in vitro culture of this orchid. Objective: The objective of the study is to establish efficient in vitro regeneration protocols for rapid mass propagation of D. thyrsiflorum. Materials and Methods: Five-month-old undehisced capsule was surface sterilized with 0.4% mercuric chloride. To study the influence of growth regulators on in vitro development of shoots and roots, the seeds obtained from the capsule were inoculated on freshly prepared Mitra medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin (KN), indole -3-butyric acid (IBA), indole-3-acetic acid (IAA), and 1-naphthyl acetic acid (NAA). Results: The KN either singly or with IAA produced more effective shooting than BAP at similar concentration and combination. Maximum shoot production (3.83 ± 0.48) was achieved in medium fortified with 1.0 mg/L KN and 2.5 mg/L IAA. IAA was the most influential among auxins tested in promoting rooting and shooting. Prominent shoot development (3.04 ± 0.73) and maximum root formation (6.52 ± 0.37) were noticed in medium supplemented with 2.0 mg/L IAA. The regenerated plantlets were subsequently hardened with 91% survival rate in greenhouse and field condition. Conclusion: The present investigation revealed the possibility of developing effective and reproducible in vitro regeneration protocols for rapid propagation of D. thyrsiflorum. The established in vitro protocols can be employed for fast regeneration of the orchid for useful phytochemical extraction without depending on the already depleted natural population.