A protocol for sample preparation before the determination in or isolation of lipids from human milk is presented. Methods for the direct determination of lipids including the creamatocrit, spectrophotometric (turbidi-metric), and enzymatic methods are discussed. Procedures for the isolation of lipids including the Roese-Gottlieb, modified Folch, and dry column extractions are compared. Finally, techniques for the separation and identification of lipid classes are described.