Efficacy and tolerability are the key criteria for a successful medication in the clinic. Therefore, a new test method to obtain selective and active lead molecules has been developed. Recently, this novel screening strategy enabled a breakthrough in drug discovery in the field of herpes viruses. Here the authors report that this assay is a generally applicable screening test, which allows not only for identifying tolerable and potent antimicrobial agents in compound libraries, but also covers all potential in vitro targets of both the pathogen and the host simultaneously. The test system mimics the smallest unit of a natural infection. Host cells are incubated in the presence of the test sample and are infected with microbes, such as viruses, bacteria, or fungi. Analogous to (lethal challenge) animal models, cell survival is determined. This assay maximizes the chances of success of anti-infective drug discovery, is sensitive, robust, time- and cost-efficient, and especially effective in optimizing screening hits to lead structures and development candidates. In addition to the minimal inhibitory concentration or dose, this test system simultaneously provides the selectivity index, a measure of tolerability in vitro. The authors propose the activity selectivity assay format as a new standard in anti-infective drug discovery and clinical development.