High-throughput screening (HTS) assays enable the testing of large numbers of chemical substances for activity in diverse areas of biology. The biological responses measured in HTS assays span isolated biochemical systems containing purified receptors or enzymes to signal transduction pathways and complex networks functioning in cellular environments. This Review addresses factors that need to be considered when implementing assays for HTS and is aimed particularly at investigators new to this field. We discuss assay design strategies, the major detection technologies and examples of HTS assays for common target classes, cellular pathways and simple cellular phenotypes. We conclude with special considerations for configuring sensitive, robust, informative and economically feasible HTS assays.