Protein phosphorylation is a key posttranslational modification, which reversibly regulates almost all processes in the living cell. Deregulated signaling is a hallmark of cancer and other diseases, and protein kinases are prominent drug targets. Phosphorylation events are commonly probed in a targeted manner by phosphorylation-specific antibodies. In contrast, advances in proteomics technology, including phosphopeptide enrichment, high-accuracy mass spectrometry, and associated bioinformatics now make it possible to analyze entire phosphoproteomes. Quantitative methods can assess the relative change in phosphorylation for several thousand sites in a single experiment. Here we review enrichment strategies and methods for mass spectrometric fragmentation and analysis of phosphopeptides. We also describe different quantitative methods and their application to problems in cell signaling and drug target discovery. Emerging phosphoproteomics technologies are becoming more comprehensive, robust, and generically applicable to a wide range of questions, including areas outside traditional eukaryotic cell signaling such as Ser/Thr/Tyr signaling in bacteria.