Analytical strategies based on NMR (H-1 and C-13), IR (infrared), and GC (gas chromatography) techniques have been developed for the molecular level characterization of Scull let and ultrasonic solvent extracts of yeast biomass samples generated on a lab scale by different yeast, feed, and diverse culture conditions, with an objective to explore biodiesel potential. The extraction efficiency of each solvent (cyclohexane, chloroform, methanol) toward extraction of neutral lipids (total glycerides (TG), free fatty acids (FFA), and polar lipids have been determined and compared with regards to the nature of fatty acid components extracted in each solvent fractions. The fatty acid composition of yeast extracts has been found to be similar to vegetable oils, mostly rich in C16:0, 18:0, and C18:N (N = 1-3) fatty acids as indicated by the combined NMR, GC, and GC-MS analyses. The analytical protocol developed has established that H-1 NMR techniques can be used directly and rapidly without any sample treatment and prior separation to determine total neutral lipid content (TG, FFA), nature of fatty acids/ester, polyunsaturated fatty esters (PUFE), iodine value, etc. NMR results of nature of unsaturated fatty acids/esters (C18:N, N = 1-3) have been validated by GC and GC-MS analyses. The results have shown the presence of C18:1 and C18:2 as the predominant unsaturated fatty acid components besides common saturated fatty acids. The content and composition of biomass has been found to be specific to types of yeast and feed used for cultivation. The NMR methods offer great potential for rapid screening of yeast for generation of yeast biomass with desired lipid content, quality, and biodiesel potential and value added PUFE, keeping in view of the cost economics of overall generation cost of the biomass.