Microbial single-cell oils (SCOs) are considered a sustainable alternative for producing biodiesel, oleochemicals, and other valuable bioproducts. The oleaginous yeast Rhodotorula glutinis is a suitable organism for lipid production due to its ability to accumulate high biomass and lipids while utilizing a wide variety of carbon sources. This study explores the use of fermentable sugars derived from agro-industrial wastes as low-cost substrates for yeast fermentation. A separate hydrolysis and fermentation approach was employed to convert agricultural residues into usable sugars that support yeast growth and lipid synthesis. Various waste-derived substrates, including lignocellulosic biomass and industrial by-products, were evaluated as alternative carbon sources to conventional glucose. The produced yeast lipids were further analyzed to assess their suitability for biofuel and biorefinery applications. Overall, the work focuses on developing a cost-effective and sustainable bioprocess for producing environmentally friendly lipids using renewable waste resources.