Lipid droplets (LD) are in the spotlight of lipid research because of the link of lipid storage to health and disease and the just incipient understanding of their involvement in cellular processes apart from nonpolar lipid metabolism. Yeast is an excellent model organism to study the lipidome and proteome of LD under different environmental conditions and to address new aspects of LD biology and chemistry. In this chapter, we describe a versatile protocol for the isolation of LD at high purity and address specific demands for handling different yeast species. Moreover, we discuss the analysis of the LD proteome and lipidome based on standard methods such as thin layer chromatography (TLC), gas liquid chromatography (GLC), mass spectrometry (MS) as well as GLC/MS. Finally, we point out similarities and disparities of LD proteome and lipidome from the three different yeasts Saccharomyces cerevisiae, Yarrowia lipolytica, and Pichia pastoris.
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