Lipids play critical roles in energy homeostasis, membrane structure, and signaling. Using liquid chromatography and mass spectrometry, we provide a comprehensive semiquantification of lipids during the life cycle of Drosophila melanogaster (230 glycerophospholipids, 210 sphingolipids, 6 sterols and sterol esters, and 60 glycerolipids) and obtain biological insights through this biochemical resource. First, we find a high and constant triacylglycerol-to-membrane lipid ratio during pupal stage, which is nonobvious in the absence of nutrient uptake and tissue remodeling. Second, sphingolipids undergo specific changes in headgroup (glycosylation) and tail configurations (unsaturation and hydroxylation on sphingoid base and fatty acyls, respectively), which correlate with gene expression of known (GIcT/CG6437; FA2H/ CG30502) and putative (Cyt-b5-r/CG13279) enzymes. Third, we identify a gender bias in phosphoethanolamine-ceramides as a lead for future investigation into sexual maturation. Finally, we partially characterize ghiberti, required for male meiotic cytokinesis, as a homolog of mammalian serine palmitoyltransferase.