Chemical genetics is an approach for identifying small molecules with the ability to induce a biological phenotype or to interact with a particular gene product, and it is an emerging tool for lead generation in drug discovery. Accordingly, there is a need for efficient and versatile synthetic processes capable of generating complex and diverse molecular libraries, and Diversity-Oriented Synthesis (DOS) of small molecules is the concept of choice to give access to new chemotypes with high chemical diversity. In this review, the combination of chemical genetics and diversity-oriented synthesis to identify new chemotypes as hit compounds in chemical biology and drug discovery is reported, giving an overview of basic concepts and selected case studies.