Serum phospholipids that derive from glycerol are hydrolyzed to diglycerides and by-products in the presence of phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). The diglycerides produced are saponified, together with the triglycerides present, to glycerol and salts of fatty acids. Measuring the glycerol produced yields a measure of the total lipid glycerol in serum, and is useful for the estimation of the phospholipid and total lipid content of serum.