Protein phosphorylation is among the most abundant post-translational modifications (PTM) in biology. Carried out by protein kinases, this enzymatic reaction plays a key role in nearly all signaling pathways. Functional annotation of phosphorylation sites in a growing number of human diseases and physiological conditions underscores the importance of protein kinases in medicine. The advent of mass spectrometry-based phosphoproteomics allows for the collection of massive amount of data, leading to an ever-increasing number of experimentally detected serine, threonine, and tyrosine phosphorylation sites. Nevertheless, linking these phosphorylation events to biological processes remains challenging and laborious, leaving a tremendous number of unannotated sites without association to any specific signaling pathway. As such, connecting phosphorylation sites with their upstream kinases is a key challenge in the phosphorylation field. Well-studied protein kinases are generally known to recognize specific amino acid residues at multiple positions surrounding the site of phosphorylation. This short linear motif, which is characteristic of a given protein kinase, ensures fidelity in signaling pathways regulating phosphorylation at a given Ser, Thr, and Tyr residues. Knowledge of kinase recognition motifs can facilitate discovery of new substrates and reveal the connection between phosphorylation events and the associated signaling cascades. However, to date, phosphorylation site sequence motifs are known for only a subset of the human protein kinome. To address this gap in knowledge, we experimentally characterize the optimal substrate specificity for nearly the entire human serine/threonine kinome and the entire tyrosine kinome. In the first part of this work, I presented the Kinase Library - a global substrate specificity atlas of the human protein kinome, and developed a prediction tool for kinase-substrate interactions and kinase activity in high-throughput phosphoproteomics data based on this atlas. In the second part of the work, I demonstrated the applications of the Kinase Library in cancer and COVID-19. First, I investigated the presentation of phosphorylated antigens by MHC-I. I explored the different features of presented phosphopeptides, and applied the Kinase Library to identify potential upstream kinases and signaling pathways from which the presented phosphorylation sites originated. Then, I examined the role of protein kinases in COVID-19. I carried out a wide phosphoproteomics characterization of cells infected with SARS-CoV-2 and utilized the Kinase Library in order to reconstruct the phosphorylation cascade of the SARS-CoV-2 nucleocapsid protein. In summary, this work represents a comprehensive substrate specificity atlas of the human protein kinome, and its applications in different biological and medical fields. This system, together with the constantly improving phosphoproteomics techniques, will allow the identification of many novel kinase-substrate interactions in different biological processes. As such, the Kinase Library provides the scientific community with a powerful tool to unravel the regulation mechanisms of many orphaned phosphorylation events and reveal yet-to-be-discovered roles of protein kinases in human diseases and life.