Hemidesmus indicus, commonly known as Indian Sarsaparilla, is a medicinal plant widely used in traditional Ayurvedic and Unani medicine due to its bioactive compounds, including tannins, sterols, and saponins. Its roots exhibit significant pharmacological properties, such as antimicrobial, anticancer, and anti-inflammatory effects. However, unsustainable harvesting and low root biomass yield pose threats to its wild populations. To support conservation and breeding efforts, this study establishes a standardized protocol for in vitro pollen germination and cryopreservation. Pollen is collected between 8:00 and 11:00 a.m. during peak anthesis from October to January. The collected pollen grains are dehydrated using zeolite granules, and viability is assessed using Brewbaker and Kwack’s medium (containing 20% sucrose, 30 mg calcium nitrate, 10 mg potassium nitrate, 10 mg boric acid, and 20 mg magnesium sulfate). Post-cryopreservation viability is evaluated by thawing pollen at 27 ± 3 °C and calculating germination percentages. This protocol offers a reliable strategy for conserving genetic diversity and improving root biomass production in H. indicus, ensuring its sustainable utilization and long-term preservation.