DiscussionPhenotypic assays are becoming increasingly important in primary hit finding due to the shift from target-based approaches using engineered cell lines towards more complex models using primary cells for physiologically relevant readouts. The use of high-content screening, and particularly live-cell imaging, enables novel assays to be created. Such assays require sophisticated, custom-made, software for the analysis of the kinetics derived from the image series; as an example the analysis of live-cell contractility will be presented. Furthermore, target hypothesis generation of hit compounds is a topic to take into account when using phenotypic assays in lead discovery. Strategies for hypothesis generation and in particular the use of HCS fingerprint analysis will be discussed.