Natural products have been extensively used to treat diseases throughout human history. These are mainly because natural products normally target biological macromolecules selectively. Target identification could help us to develop new therapeutic agents and discover new biological pathways underlying human diseases. Herein, we highlight some recent examples of using natural products and their derivatives as chemical probes to identify the molecular targets and elucidate mode of action. Natural products have been extensively used to treat diseases throughout human history. These are mainly because natural products normally target biological macromolecules selectively. Target identification could help us to develop new therapeutic agents and discover new biological pathways underlying human diseases. Herein, we highlight some recent examples of using natural products and their derivatives as chemical probes to identify the molecular targets and elucidate mode of action.