The 512 protein kinases encoded by the human genome are a prime example of nature's ability to create diversity by introducing variations to a highly conserved theme. The activity of each kinase domain is controlled by layers of regulatory mechanisms involving different combinations of post-translational modifications, intramolecular contacts, and intermolecular interactions. Ultimately, they all achieve their effect by favoring particular conformations that promote or prevent the kinase domain from catalyzing protein phosphorylation. The central role of kinases in various diseases has encouraged extensive investigations of their biological function and three-dimensional structures, yielding a more detailed understanding of the mechanisms that regulate protein kinase activity by conformational changes. In the present review, we discuss these regulatory mechanisms and show how conformational changes can be exploited for the design of specific inhibitors that lock protein kinases in inactive conformations. In addition, we highlight recent developments to monitor ligand-induced structural changes in protein kinases and for screening and identifying inhibitors that stabilize enzymatically incompetent kinase conformations.