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 regulated pathways remain a bottleneck in the drug R&D processes. Fortunately, chemical proteomic strategies based on the perfect combination of various targeted affinity chromatography and high-throughput MS analysis have emerged as a powerful tool for the large-scale identification of proteome-wide drug–protein interactions, and demonstrated great promise in elucidating complex underlying mechanisms of drug action against cancers. In this context, an updated overview of the chemical proteomic strategies, such as activity-based protein profiling (ABPP), compound-centric chemical proteomics (CCCP), and other targeted affinity chromatographic approaches for modern anticancer drug discovery and development will be provided. Some most recent successful applications in this area will be highlighted. Future perspectives on this subject will also be discussed with a particular emphasis on small molecule natural products and their derivatives.