Abstract
Monitoring how, when, and where small molecules engage their targets inside living cells is a critical step in chemical biology and pharmacological research, because it enables compound efficacy and confirmation of mode of action to be assessed. In this mini-review we summarize the currently available methodologies to detect and prove direct target engagement in cells and offer a critical view of their key advantages and disadvantages. As the interest of the field shifts toward discovery and validation of high-quality agents, we expect that efforts to develop and refine these types of methodologies will also intensify in the near future.