Cancer is one of the leading causes of death globally. Drug discovery and development against cancer is thus among the most pursuing goals nowadays. Although the majority of anticancer drugs targeted on proteins, the identification and validation of drug targets and their regulatedpathways remain a bottleneck in the drug R&D processes. Fortunately, chemical proteomicstrategies based on the perfect combination of various targeted affinity chromatography andhigh-throughput MS analysis have emerged as a powerful tool for the large-scale identificationof proteome-wide drug–protein interactions, and demonstrated great promise in elucidatingcomplex underlying mechanisms of drug action against cancers. In this context, an updatedoverview of the chemical proteomic strategies, such as activity-based protein profiling (ABPP),compound-centric chemical proteomics (CCCP), and other targeted affinity chromatographicapproaches for modern anticancer drug discovery and development will be provided. Somemost recent successful applications in this area will be highlighted. Future perspectives on thissubject will also be discussed with a particular emphasis on small molecule natural productsand their derivatives.