Protein kinase inhibitors are an increasingly important class of targeted anticancer therapeutics. More than two dozen new drugs of this class have entered routine clinical use over the past decade. This review article focuses on how the development of methods to study the kinome- and proteome-wide selectivity of kinase inhibitors, in conjunction with advances in the structural understanding of kinase inhibitor binding modes, has resulted in a better appreciation of the mechanism of action of clinical kinase inhibitors. I provide examples of how this has led to the discovery of unexpected off-target effects, intriguing cases in which kinase inhibitors may cause pathway activation, and new mechanisms responsible for resistance to kinase inhibitors. Finally, I illustrate that although certain kinase targets may be pharmacologically easily tractable, a better understanding of the regulation and biology of the targets is required to generate drugs that are efficacious in cancer patients. © 2014 American Chemical Society.