Chemical probes represent versatile tools to validate disease-modifying targets. However, evaluating the selectivity of chemical probes in complex cellular systems is a major challenge that needs to be addressed to better understand the mode of action of small molecules and the interpretation of their pharmacological effects. Chemoproteomics has emerged as a key technology to characterize the mode of action of pharmacological modulators such as chemical probes and drugs, and these studies have unraveled the cellular targets of many bioactive compounds. Here we review the role of chemical probes for the validation of new therapeutic targets and their characterization by proteome wide affinity- and activity-based chemical proteomics and recently developed label-free technologies. Development of highly selective chemical probes has pioneered new target areas and validated new disease-modifying drug targets. Chemoproteomics offers a proteome-wide evaluation of the selectivity of chemical tools, minimizing the risk that undetected off-targets lead to observed pharmacological responses. Mass spectrometry–based methods also lead to a better understanding of the mode of action of approved drugs, including some of the observed side effects. Thus, chemoproteomics has developed into a key technology for early- as well as late-stage drug development.