This chapter discusses the process of mapping phosphorylation sites in proteins by mass spectrometry (MS). MS is ideally suited for the direct identification of protein phosphorylation sites. Phosphopeptides present in mixtures can be sequenced at the femtomole level without the need for extensive purification. A variety of mass spectrometry-based approaches have been employed to map phosphorylation sites in proteins. A great advantage of these methods is that they do not require prior labeling of the target protein with 32p. Thus, it is possible to isolate rare proteins from large-scale cultures for phosphopeptide mapping studies. Regardless of the method used to map phosphorylation sites, it is imperative that the native phosphorylation state of the target protein be preserved during isolation. The chapter describes general strategies for isolating phosphoproteins from budding yeast cells by drawing reference to two specific examples: the S-Cdk inhibitor, Sic1, and the mitotic exit inhibitor, Net1. The chapter presents an overview of the mass spectrometric methods that are used to map specific phosphorylation sites in these proteins.