Catharanthus roseus (L.) G.Don, a medicinally valuable plant, faces regeneration challenges due to its unique floral structure, necessitating artificial pollination. Effective conservation and breeding require optimal pollen collection, viability assessment, and long-term storage. This study employed in vitro viability tests using Brewbaker and Kwack’s medium mixed with pollen and dispersed in a hanging drop position on a cavity slide. The slide was then incubated in a moisture chamber, optimizing pollen tube growth and germination. Catharanthus roseus conservation is challenged by its unique floral structure. Pollen cryopreservation ensures germplasm preservation and facilitates breeding. This study evaluates pollen viability, cryopreservation, and fertility, providing a simplified protocol for effective conservation and medicinal research.