High-throughput screening (HTS) is a production process aimed at testing tens of thousands of compounds per hour for the identification of a small subset of compounds that display a desired property, such as affecting a biological target of interest. As with any production process, the output of HTS depends profoundly on the quality of input materials, assay reagents and chemical compound libraries, as well as on the reliability of the process itself, a major component of which is a biological assay employed for testing compounds.

The utility of HTS assays depends on the correlation between biological properties of model systems, macromolecules or cellular pathways, and measurable biophysical properties gauging the effect of screened compounds on the HTS target of interest. Compounds with desired properties, identified as actives or HTS hits, need to be unequivocally distinguished from “inactive” compounds. Because of the number of compounds simultaneously tested in HTS, screening assays must be extremely reproducible and robust to provide statistically reliable results. Furthermore, HTS assays must be fine-tuned to enhance their ability to distinguish hits with desired pharmacologic properties apart from all other compounds. Achieving these generic and specific traits of HTS assays is described in detail in this chapter.