Dairy phospho- and sphingolipids are gaining interest due to their nutritional and technological properties. A new HPLC method, using an evaporative laser light-scattering detector, was developed, which enabled excellent separation of glucosylceramide, lactosylceramide, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine in less than 21 min, including the regeneration of the column. No loss of column performance was observed after 1500 runs because an acid buffer was used. The output signal of the evaporative laser light scattering detector was highly dependent of the flow of the carrier gas and the temperature of the nebulizer, and was maximized by means of a response surface experimental design. Finally, raw milk, cream, butter, buttermilk, Cheddar whey, quarg, and Cheddar cheese were analyzed for their polar lipid content. The absolute values varied substantially (0.018 to 0.181 g/100 g of product). Significant differences were found in the relative content of each polar lipid class among the analyzed products.