Phosphoinositides are critically important for numerous cellular signaling pathways such as membrane trafficking, cytoskeleton rearrangement, and ion channel regulation in eukaryotic organisms. The physiological relevance of phosphoinositide metabolism at the Drosophila neuromuscular junction (NMJ) has been illustrated using several mutants that lack crucial factors of phosphoinositide signaling. Although several decades of research in both in vitro and in vivo models have led to an understanding of the mechanisms of lipid-protein interactions and downstream signaling, the details on how their temporal and spatial distribution is regulated at the sub-cellular level in vivo remains poorly understood. To obtain a better understanding of phosphoinositide signaling, detailed biochemical and cell biological approaches can best be combined with genetics. In this review, we present an overview of the methodologies available in the fruit fly Drosophila melanogaster, to genetically dissect the complex regulation of signaling pathways involving phosphoinositides.