Lipids play a central role in numerous biological events, ranging from normal physiological processes to host−pathogen interactions. The proposed semiconstant-time ³¹P,¹H−COSY NMR experiment provides identification of known and structural characterization of unknown phospholipids in complex membrane extracts with high sensitivity, based on the combination of their ¹H and ³¹P chemical shifts and coupling patterns. Furthermore, the spectra allow quantification of phospholipid composition. Analysis of the phospholipid composition of Helicobacter pylori, the causative agent of peptic ulcer disease, showed the presence of uncommon phospholipids. This novel NMR approach allows the study of changes in membrane composition in response to biological stimuli and opens up the possibility of identifying soluble phosphorus species in a number of research fields.