An improved HPLC-ELSD method has been developed for the analysis of the lipid classes of buttermilk and milk from different species, focused in the phospholipids fraction without a prior fractionation step and in a single run. The total lipid profile analysis showed the major and minor lipid compounds as cholesterol esters, triacylglycerides, cholesterol, diacylglycerides, free fatty acids, monoacylglycerides, and also the polar compounds as glucosylceramide, lactosylceramide, phosphatidyl-ethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine. The identification and quantification of the different compounds, using calibration curves made with individual standards and the low coefficients of variation obtained in the inter- and intra-assays showed the suitability of the developed method. In this study, we optimized and validated a quantitative HPLC-ELSD method at a concentration level suitable for routine analysis of the major lipid classes in milk and dairy products.