Pteridophytes, an ancient and diverse group of vascular plants, are distinguished by their spore-based reproduction and remarkable adaptability to a wide range of habitats, from tropical rainforests to arid regions and alpine zones. These plants play a crucial role in ecosystem functions such as nutrient cycling, soil stabilization, and habitat formation for other species. Their ability to disperse spores over long distances facilitates colonization in remote areas, enhancing their survival in diverse environmental conditions. However, many pteridophyte species face increasing threats due to habitat loss, climate change, invasive species, and other anthropogenic pressures, highlighting the urgent need for targeted conservation strategies. Traditional seed banking methods are unsuitable for ferns due to their reliance on spores for reproduction. In this context, cryopreservation offers a promising long-term conservation strategy, allowing spores to be stored at ultra-low temperatures while maintaining viability. This approach also prevents genetic drift and degeneration over time. This chapter presents optimized cryopreservation protocols for the spores of three ecologically significant species: Pteris vittata L., Asplenium nidus L., and Nephrolepis cordifolia (L.) C. Presl. These species were selected for their ecological importance, horticultural value, and potential in bioremediation. The chapter details methodologies for spore collection, sterilization, desiccation, storage, and post-storage viability assessment. The findings contribute to the advancement of scalable and effective preservation techniques, supporting the long-term conservation of pteridophytes and their ecological roles.