Recent advances have expanded the role of lipid droplets (LDs) beyond passive lipid storage, implicating their involvement in various metabolic processes across mammalian tissues. Neuronal LDs, long debated in existence, have been identified in several neural structures, raising questions about their contribution to neurodegenerative disorders. Elucidating the specific chemical makeup of these organelles within neurons is critical for understanding their implication in neural pathologies. However, the inherent submicron size and low abundance in neurons have posed challenges in their isolation; and physical and biochemical characterization. To overcome this obstacle, we blocked neuron-specific triglyceride lipase to enlarge LDs, rendering them amenable to floatation in a sucrose gradient. We further employed an improved methodology of lipidomic analysis on the LDs purified from cultured primary neurons, offering insights into their unique lipid-protein composition. Future integration of this method with high-throughput techniques holds promises for uncovering disease-specific alterations in lipid metabolism, providing new avenues for potential therapeutic interventions.