We designed and constructed an antibiotic screening system by using antibiotic responsive genes as reporters. Plasmid pCS26 carrying a promoterless luminescence reporter, luxCDABE, was used as the vector and the promoter regions of antibiotic responsive genes/operons from Escherichia coli were cloned upstream of the lux reporter to form the first part of the screening reporter array. Random promoter library of Salmonella enterica and Pseudomonas aeruginosa were screened for antibiotic responsive clones which consist of the second part of the screening array. The selected final reporter array responded to different antibiotics in distinct patterns and enabled in vivo high-throughput screening for antibiotics. Unknown antibiotics could, in general, be classified by analyzing the response patterns. This screening system is both sensitive and efficient and should prove to be a useful tool for screening new antibiotic compounds.