Dendrobium spectabile (Blume) Miq. is one of Papuan orchids that is of economic value and threatened. At present, the optimal protocol for asymbiotic seed germination and planlet development of D. spectabile is not yet available. This research aimed to develop an optimal and comprehensive protocol for in vitro culture of D. spectabile to support the conservation and reintroduction of this species. The experiment was conducted using a completely random design, including seed sowing, protocorm subculture (subculture 1), plantlet subculture (subculture 2), and plantlet acclimatization. The highest germination rate at 3 MAS (months after sowing) was found in modified Knudson's C (92.59%). The first subculture at 4 MAP (months after planting) showed that the highest leaf and root growths were found in modified Vacin and Went (4.12 and 2.13, respectively). The second subculture at 6 MAP showed that the highest leaf growth was found in full strength of Murashige and Skoog supplemented with 100 g/l banana homogenate (5.49), while the highest number of roots and average root extension were found in half strength of Vacin and Went supplemented with 100 g/l banana homogenate (7.05 and 0.47 cm, respectively). The plantlets were best acclimatized in media consisting of tree fern fibre and sphagnum moss (2:1).