The (poly-)pharmacological activities of a drug can only be understood if its interactions with cellular components are comprehensively characterized. Mass spectrometry-based chemical proteomics approaches have recently emerged as powerful tools for the characterization of drug-target interactions in samples from cell lines and tissues. At the same time, off-target activities can be identified. This information can contribute toward optimization of candidate drug molecules and reduction of side effects. In this review, we describe recent advances in chemical proteomics and outline potential applications in drug discovery.