This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and aminoacylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model "universal" mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a specific target of an antibiotic within the translational pathway of prokaryotic cells. © 2007 Elsevier Inc. All rights reserved.