The article discusses the importance of target identification and mechanism of action studies in chemical biology and drug discovery. It highlights the use of cell-based assays to discover small molecules with biological activity, and the need for follow-up studies to determine the precise protein targets responsible for observed phenotypes. Target identification can be achieved through direct biochemical methods, genetic interactions, or computational inference. The article emphasizes the integration of multiple approaches to fully characterize on-target and off-target effects of small molecules. Modern chemical biology and drug discovery aim to identify small molecules that modulate the functions of target proteins. Historically, natural sources have been important for such molecules, but recent advances have shifted towards screening large compound collections for desired biological responses. This includes both natural and synthetic small molecules. The article also discusses the shift from purified protein-based assays to cell- or organism-based phenotypic assays, which preserve the cellular context of protein function. The cost of this shift is that the precise protein targets or mechanisms of action remain to be determined. Even after a relevant target is established, additional functional studies may help identify off-target effects or establish new roles for the target protein in biological networks. The article reviews various approaches to target identification, including direct biochemical methods, genetic interaction methods, and computational inference methods. It also discusses the challenges and limitations of each approach, as well as the importance of integrating multiple methods for accurate target identification. The article highlights the use of affinity purification, genetic interaction, and computational methods to identify protein targets of small molecules. It also discusses the role of proteomics and bioinformatics in target identification, as well as the importance of unbiased approaches in screening. The article concludes that a combination of approaches is most likely to yield accurate results, and that the integration of data from multiple sources is essential for understanding the mechanisms of action of small molecules.The article discusses the importance of target identification and mechanism of action studies in chemical biology and drug discovery. It highlights the use of cell-based assays to discover small molecules with biological activity, and the need for follow-up studies to determine the precise protein targets responsible for observed phenotypes. Target identification can be achieved through direct biochemical methods, genetic interactions, or computational inference. The article emphasizes the integration of multiple approaches to fully characterize on-target and off-target effects of small molecules. Modern chemical biology and drug discovery aim to identify small molecules that modulate the functions of target proteins. Historically, natural sources have been important for such molecules, but recent advances have shifted towards screening large compound collections for desired biological responses. This includes both natural and synthetic small molecules. The article also discusses the shift from purified protein-based assays to cell- or organism-based phenotypic assays, which preserve the cellular context of protein function. The cost of this shift is that the precise protein targets or mechanisms of action remain to be determined. Even after a relevant target is established, additional functional studies may help identify off-target effects or establish new roles for the target protein in biological networks. The article reviews various approaches to target identification, including direct biochemical methods, genetic interaction methods, and computational inference methods. It also discusses the challenges and limitations of each approach, as well as the importance of integrating multiple methods for accurate target identification. The article highlights the use of affinity purification, genetic interaction, and computational methods to identify protein targets of small molecules. It also discusses the role of proteomics and bioinformatics in target identification, as well as the importance of unbiased approaches in screening. The article concludes that a combination of approaches is most likely to yield accurate results, and that the integration of data from multiple sources is essential for understanding the mechanisms of action of small molecules.