Much of the current research on lipids points to their role in regulating the infectious disease process of pathogenic microorganisms. This is particularly important in the case of sterols, which vary among the major pathogenic fungi and contribute differentially to their susceptibility to antifungals. Unfortunately, studies of such pathogenic fungi are limiting, from incomplete lipid extraction and analysis. In addition to challenges posed by mutant collections of pathogenic fungi, including Cryptococcus neoformans and Candida spp., fungal characteristics, such as encapsulation or biofilm formation, further complicate studies. This chapter outlines successful modifications made to traditional methods for such fungal pathogens.