Ashwagandha (Withania somnifera L.), an important medicinal herb used in Ayurvedic, Unani, and Homeopathic medicine, is valued for its pharmacological benefits, such as anti-aging, cognitive enhancement, and anti-inflammatory properties. Its active compounds, including withanolides and withaferins, are primarily found in its roots. Despite its growing global demand, Ashwagandha cultivation faces challenges like low yields, pest susceptibility, and inconsistent phytochemical content. To address these issues, this study developed a comprehensive protocol for pollen cryopreservation aimed at breeding resilient varieties. Pollen was collected during peak anthesis (9–11 am) in the flowering season (April–June), followed by dehydration using zeolite granules in a desiccator to achieve a moisture content of 10–12%. The pollen was germinated in vitro using Brewbaker and Kwack’s medium, consisting of 15% sucrose, calcium nitrate (300 ppm), potassium nitrate (100 ppm), boric acid (100 ppm), and magnesium sulphate (200 ppm). The pollen was cryopreserved in liquid nitrogen after dehydration, with viability tested post-storage using the hanging drop technique and Alexander’s stain. This protocol ensures the availability of high-quality pollen for breeding stress-tolerant, high-yield Ashwagandha varieties, supporting the growing global demand.