Most mammalian proteins are transiently phosphorylated on seryl, threonyl, or tyrosyl positions by kinases and dephosphorylated by phosphatases in response to specific intra- or extracellular signals. Many diseases, such as cancer, are caused by altered kinase and phosphatase activities or changed expression levels of either of these enzymes. Thus phosphopeptides are important and universal tools to study disease-specific changes, such as protein-protein/DNA interactions or hyperactive kinases, using phosphopeptides or the corresponding mimics. Here we describe two generally applicable techniques to synthesize multiply phosphorylated peptides as basic tools for drug development. The phosphopeptides were purified to homogeneity and characterized by mass spectrometry.