Docosahexaenoic acid (DHA, 22:6 n-3) is an omega-3 fatty acid with a 22 carbon acyl chain containing six cis double bonds and is predominantly found in membrane glycerophospholipids. Dietary consumption of DHA has been positively linked with the prevention of numerous pathologies and consequently, it has been the focus of extensive research over the last four decades. Nevertheless, our understanding of its molecular mode of action is not well understood. One likely mechanism is through DHA’s influence on cell membranes and the proteins embedded within them. This influence may be altered depending on the glycerophospholipid head group DHA is esterified to and its fatty acid partner, i.e., the specific glycerophospholipid molecule. Accordingly, an understanding of the exact glycerophospholipid distribution of DHA within a tissue is important if we wish to gain further insight into its role in the prevention of disease. In this chapter a rapid, shotgun lipidomic approach for identifying the molecular glycerophospholipid distribution of DHA is described.