Mass spectrometry-based phosphoproteomics is currently the leading methodology for the study of global kinase signaling. The scientific community is continuously releasing technological improvements for sensitive and fast identification of phosphopeptides, and their accurate quantification. To interpret large-scale phosphoproteomics data, numerous bioinformatic resources are available that help understanding kinase network functional role in biological systems upon perturbation. Some of these resources are databases of phosphorylation sites, protein kinases and phosphatases; others are bioinformatic algorithms to infer kinase activity, predict phosphosite functional relevance and visualize kinase signaling networks. In this review, we present the latest experimental and bioinformatic tools to profile protein kinase signaling networks and provide examples of their application in biomedicine.