Polar glycerolipids are a diverse family of lipid molecules that form the bulk of bacterial and eukaryotic microbial membranes. The earth and ocean sciences has a long history of using fatty acids as biomarkers for microbes, but have only recently begun to examine the intact polar lipids from which they are derived. Current analytical approaches rely on laboriously quantifying the molecular ions of each of these species independently. Thus, we saw a need for a method for quantifying polar glycerolipid classes that was: (i) selective for individual classes, (ii) inclusive of all species within a class, (iii) independent of foreknowledge of the molecular ions of the polar glycerolipid, and (iv) amenable to automated, high-throughput data analysis methods. Our new HPLC-electrospray-ionization triple-quadrupole MS (HPLC-ESI-TQMS) method can be applied to quantify the nine major classes of polar glycerolipid in planktonic communities: the phospholipids phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine; the glycolipids monoglycosyldiacylglycerol, diglycosyldiacylglycerol and sulfoquinovosyldiacylglycerol; and the betaine lipids diacylglyceryl trimethyl homoserine, diacylglyceryl hydroxymethyl trimethyl-β-alanine, and diacylglyceryl carboxyhydroxymethylcholine. The analyses rely on neutral loss and parent ion scan events that yield one chromatogram for each class of polar glycerolipid, simplifying downstream analysis and increasing sample throughput. The efficacy of the method was demonstrated by analyzing plankton community samples from a variety of marine environments.