Protein phosphorylation, one of the most ubiquitous post-translational modifications of proteins, is reversibly controlled by protein kinases and phosphatases, and regulates a variety of cell functions through cellular signal transduction pathways. These signals are involved in various diseases such as cancer, and often cause the disease itself or its progression. Phosphoproteomics has been explored to analyze protein phosphorylation comprehensively and has been applied to monitoring the entire network of cellular signal transduction. In this review, the frontlines of phosphoproteomics as well as its application to cancer molecular targeting drug discovery, therapy and diagnostics are described.