In spite of some progress in understanding the molecular basis of lipid-associated disorders, major questions about the regulation of synthesis and degradation of lipids and the interaction of these processes with other aspects of cellular physiology are still unresolved. Studies in reference organisms such as various yeast species, the fruit fly Drosophila melanogaster, or the nematode Caenorhabditis elegans complement efforts in mouse models as well as clinical studies in humans to address these questions. Imaging techniques play a pivotal role in understanding lipid droplet biology, and the implementation of imaging-based high-content screens of mutant collections has led to the identification of novel molecular players. This study focuses on novel fluorescent probes as well as spectroscopic imaging techniques to investigate lipid droplet formation and turnover in yeast. The application and limitations of such techniques in understanding lipid storage and turnover are discussed.