The article discusses the importance of target identification and mechanism-of-action studies in chemical biology and drug discovery. It highlights the increasing use of cell-based assays to discover new biologically active small molecules, which allows for testing their effects in disease-relevant settings. However, these studies often require follow-up to determine the specific protein targets responsible for observed phenotypes. The article outlines three main approaches for target identification: direct biochemical methods, genetic interactions, and computational inference. Each method has its strengths and limitations, and a combination of these approaches is often necessary to fully characterize on-target and off-target effects. The article also emphasizes the importance of integrating multiple approaches to gain a comprehensive understanding of the molecular mechanisms of small molecules. Recent advances in techniques such as affinity purification, quantitative proteomics, and computational modeling are discussed, along with examples of successful integrations of different methods to determine the mechanism of action of small molecules. The article concludes by emphasizing the value of integrating large sets of disparate data to understand the molecular mechanisms of small molecules in biological systems.The article discusses the importance of target identification and mechanism-of-action studies in chemical biology and drug discovery. It highlights the increasing use of cell-based assays to discover new biologically active small molecules, which allows for testing their effects in disease-relevant settings. However, these studies often require follow-up to determine the specific protein targets responsible for observed phenotypes. The article outlines three main approaches for target identification: direct biochemical methods, genetic interactions, and computational inference. Each method has its strengths and limitations, and a combination of these approaches is often necessary to fully characterize on-target and off-target effects. The article also emphasizes the importance of integrating multiple approaches to gain a comprehensive understanding of the molecular mechanisms of small molecules. Recent advances in techniques such as affinity purification, quantitative proteomics, and computational modeling are discussed, along with examples of successful integrations of different methods to determine the mechanism of action of small molecules. The article concludes by emphasizing the value of integrating large sets of disparate data to understand the molecular mechanisms of small molecules in biological systems.