Abstract: Background and Objective: Micro-propagation technique significantly improves the quality of the potentially important crops. In this study in vitro propagation of Amomum subulatum through sprouting bud was carried out to develop a suitable protocol for development of superior quality plantlets. Materials and Methods: Sprouting buds were sterilized and cultured into MS medium supplemented with different plant growth regulators. The data presented were average of the triplicate with standard deviation. Results: Augmentation of Murashige and Skoog (M.S.) medium with 2.0 mg L–1 BAP (6-benzylaminopurine) recorded the highest number of shoot proliferation (8.07±0.06 shoots/explant) followed by AS 3 (1.5 mg L–1 BAP) with 6.23±0.02 shoots/explant, AS 10 with 5.12±0.02 and AS 7 (0.8 mg L–1 BAP+0.2 mg L–1 IAA) with 5.09±0.09 shoots. M.S. media AsR5 supplemented with 1 mg L–1 IBA+0.2 mg L–1 IAA has highest root proliferation with 7.79±0.11 roots/shoot. Media AsR4 (0.8 mg L–1 IBA+0.2 mg L–1 IAA) has 60.06±0.15 root/shoot followed by media AsR3 (0.6 mg L–1 IBA+0.2 mg L–1 IAA) with 4.14±0.04 roots/shoot. The maximum percentage of acclimatization, hardening and rhizomes production of in vitro derived plants in shad net house conditions was between 75-90%. Conclusion: The present study revealed that, highest growth of plantlets in terms of shoots developed/explant and number of leaves developed/shoots was achieved in MS media having BAP with the concentration of 2.0 mg L–1. Likewise, 1.0 mg L–1 BAP with 0.2 mg L–1 of IAA also exhibit positive impact on root proliferation and root length. The protocol developed could be used for developing the superior quality of plants.