Publisher Summary The chapter presents a discussion on extraction of phospholipids and analysis of phospholipid molecular species. The phospholipids (PLs) are a family of molecules that, with the exception of sphingomyelin (SM), are characterized by the presence of a phosphate group esterified to the sn -3 position of glycerol and 1 (lyso-PLs) to 4 (cardiolipin) acyl residues attached through an ester bond at the sn -2 position of glycerol and either an ester (acyl), ether (alkyl), or vinyl ether (alkenyl) bond at the sn -1 position of glycerol. The separation of a particular PL class into its various molecular species can be readily accomplished by reversed-phase chromatography of either the intact PL or after the PL has been converted to an appropriate derivative. There are advantages and disadvantages to both procedures. The first step in the fractionation of PLs is to extract the lipids from the tissue. This is accomplished by extracting the tissue with a combination of organic solvents: (1) the lipids are chemically stable, (2) any enzymes that might metabolize the lipids are denatured, and (3) where miscibility with water is sufficient so that a single phase is formed (to ensure an efficient extraction).