The genomics revolution has resulted in a vast amount of knowledge about human physiology and how different diseases affect function. Additional technological advancements, such as transcriptomics and proteomics have pushed insights even further. However, translating these findings into new drugs has progressed less rapidly. One of the bottlenecks is the challenge of going from the identity of the most appropriate cellular target, to having a drug, usually a small molecule, that affect the biology of the target in a manner that corrects the disease phenotype. A type of strategy developed to address this challenge is high-throughput screening (HTS). HTS is used to identify small molecules that bind a target of interest, leading to desired change in target’s behavior (usually activity) and/or to cellular phenotype of interest. These methods commonly use robotic sample handling, small volumes, rapid data acquisition, and sophisticated software and compound library management systems, to conduct thousands, even millions of small scale experiments and identify the most promising hit compounds. Moreover, high-throughput methods to validated the hits in terms of their binding affinities, selectivity, in-cell target engagement, pharmacokinetic and pharmacodynamic properties, reactivity, and stability are also on the rise. This methods collection captures some of the work in this area, and it is certain to grow further as new advancements in HTS methods continue to be reported. It is a resource for anyone interested in drug discovery and development, as well as hit identification and validation