The rapid spreading of multi-drug-resistant human-pathogenic bacteria in industrialized countries and the thereto related increased morbidity and mortality urgently require decisive countermeasures to contain this imminent threat: (i) Discovery of novel anti-infectives has to outperform the emergence of novel resistance mechanisms, and the use of clinically licensed antibiotics in stock farming will have to be restricted or banned. Both of these key actions require highly sensitive and rapid technology to discover and validate novel antibiotic lead structures as well as detect trace amounts of illegal antibiotics in food samples including milk or meat. To increase the discovery rate for novel anti-infective lead structures, we have designed the Mammalian Antibiotic Sensor Technology (MAST), a mammalian cell-based screening platform with integrated cytotoxicity and bioavailability assessment of novel antibiotic structures. The basis of MAST are antibiotic biosensors derived from prokaryotic transcriptional regulators, which are highly responsive to potent antibiotic core structures of a desired class. In order to enforce the ban of certain antibiotics in food samples we developed an in vitro Biosensor ImmunoAssay (BIA) for which we have engineered prokaryote-derived biosensors into a cell-free test system for rapid and sensitive detection of antibiotics in biological samples like milk or serum.