Lipids play important roles in biological systems; they store energy, play a structural role in the cell membrane, and are involved in cell growth, signal transduction, and apoptosis. Phospholipids (PLs) in particular have received attention in the medical and lipidomics research fields because of their involvement in human diseases such as diabetes, obesity, atherosclerosis, and many cancers associated with lipid metabolic disorders. Here I review experimental strategies for PL analysis based on nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MSn). In particular, discussed are lipid extraction methods, nanoflow LC separation of PLs, effect of ionization modifiers on the ESI of PLs, influence of chain lengths and unsaturation degree of acyl chains of PLs on MS intensity, structural determination of the molecular structure of PLs and their oxidized products, and quantitative profiling of PLs from biological samples such as tissue, urine, and plasma in relation to cancer and coronary artery disease.