A wealth of information related to lipid metabolism and signaling has been revealed in recent years using mass spectrometric-based lipidomics methods. Although quantitatively sensitive, these compositional profiling methods rely on conventional tissue extractions of total lipids which results in a loss of original cellular context of lipid metabolites. We described the development of direct organelle mass spectrometry (DOMS), a high resolution MS profiling method providing the capability to directly visualize, extract, and analyze the lipid compositions in single, individual lipid droplets (LDs) from plant tissues. DOMS of lipid droplets isolated from mature cotton embryos demonstrated a surprising lipid droplet-to-droplet variability in triacylglycerol (TAG) composition that would have been concealed through conventional profiling methods and might be important for the understanding of LD biogenesis in oilseeds. Additional applications directed toward the identification of lipid and protein compositions of other organelles could have a significant impact on our general understanding of metabolism and suggest new ideas about how cells coordinate the functions of their subcellular compartments.