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Objective: To develop an efficient reproducible protocol for a critically endangered f, Pteris tripartita (P. tripartita) Sw. through spore culture. Methods: The spore derived gametophytes were grown in a half strength MS medium which sub-cultured after three months to MS medium containing growth regulators like BAP, Kinetin and GA3 with 3% sucrose. Results: The highest mean number of sporophytes (64.40) induced with 4.16 cm of length and also formed 1.51 cm of root length at 4 mg/L of BAP. On MS medium supplemented with KIN, 56.03 mean numbers of sporophytes with 2.61 cm along with 1.39 cm length of root were obtained in 4 mg/L of KIN. And, the highest mean numbers of juvenile sporophytes (59.66) were induced with 2.64 cm mean lengths of sporophytes with 2.23 cm of root length in 4 mg/L of GA3 in the MS culture medium. The in vitro developed plantlets were washed with distilled water and grown in small cups containing sterilized soil, sand and vermiculite (1:1:1). The acclimatized plantlet showed 90% of a survival rate. Conclusions: In this study, reproducible protocol could be useful for their rehabilitation and also to reduce the pressure on natural population in Asian habitat.