The ‘discovery’ of lipid droplets as a metabolically highly active subcellular organelle has sparked great scientific interest in its research in recent years. The previous view of a rather inert storage pool of neutral lipids—triacylglycerol and sterols or steryl esters—has markedly changed. Driven by the endemic dimensions of lipid-associated disorders on the one hand, and the promising biotechnological application to generate oils (‘biodiesel’) from single-celled organisms on the other, multiple model organisms are exploited in basic and applied research to develop a better understanding of biogenesis and metabolism of this organelle. This article summarizes the current status of LD research in yeast and experimental approaches to obtain insight into the regulatory and structural components driving lipid droplet formation and their physiological and pathophysiological roles in lipid homeostasis.