Comprehensive identification of quantitative changes in protein phosphorylation using mass spectrometry is becoming a common tool in cell signaling studies. To date, most of these kinase network studies are conducted in stable cancer cell lines, yeasts, or other models that are not representative of cardiovascular disease. We describe methods based on phosphopeptide enrichment after tryptic digestion of cell lysates to study changes in protein phosphorylation of endothelial cells. We used this approach to study the activation of aortic endothelial cells by oxidized phospholipids, compounds important in atherosclerosis and other inflammatory diseases.