Signaling pathways driven by protein and lipid kinases are altered in most human diseases. Therefore, pharmacological inhibitors of cell signaling are one of the most intensively pursued therapeutic approaches for the treatment of diseases such as cancer, neurodegeneration, and metabolic syndromes. Phosphoproteomics is a technique that measures the products of kinase activities and, with the appropriate bioinformatics techniques, the methodology can also provide measures of kinase pathway activation and network circuitry. Hence, due to recent technological advantages, LC-MS-based quantitative phosphoproteomics provides relevant information for the design and implementation of kinase inhibitor based therapies. Here, we review how phosphoproteome profiling is being used in translational research as a means to identify drug targets and biomarkers for personalizing therapies based on kinase inhibitors.