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Each year the number of resistant bacterial strains increases, so finding new antibiotics is one of the principal goals of modern biochemistry. Both natural extracts and chemically synthetized compounds can be used in high-throughput screening of antimicrobial agents. These methods of screening are widely used and usually new inhibitors of growth are found, but understanding the mechanism of action is a more sophisticated task. The majority of known antibiotics affect translation. Based on the tryptophan attenuator trpL and Katushka2S fluorescent protein a broad-specificity system for detection of translation inhibitors was developed. At the same time Red fluorescent protein was cloned under sulA promoter and become dependent from presence of DNA-damaging antibiotics. The approach was optimized to test up to 586 compounds per single agar plate, without the need in any enzymatic substrates and serial dilutions. By means of this method, we tested 50 thousand individual chemical compounds for antibacterial activity and the mechanisms of action. Several new translation inhibitors were found. Their MOA was confirmed by in vitro experiments.