Sclerospora graminicola is a plant pathogen that causes downy mildew in pearl millet, foxtail millet, and maize worldwide. It affects the yield and quality of crops by disrupting chlorophyll biosynthesis and photosynthesis. Fungal transformation provides an excellent approach to genetic manipulation involving protoplasts. Scientists till date have used protoplasts for transformation studies. Intra and inter specific hybridization of fungal species have been reported for strain improvement and fungicide resistance in many fungal strains. Firstly, we studied the optimization method of protoplast preparation of Sclerospora graminicola and found various factors that may affect the yield during protoplast preparation, such as mycelial stages, pH values, and osmotic stabilizers. Secondly, in the protoplasts isolated, we explored the effects of various osmotic conditioners on the protoplasts viability. The results showed, a protocol has been developed to isolate protoplasts from the infective propagules viz., sporangia, zoospores, oospores and mycelia of S. graminicola using commercially available enzyme NOVOZYME-249 possessing pectinase and proteinase activity. The method standardized for liberation of protoplasts is rapid, reliable and high yielding.