Aminoacyl-tRNA synthetases are enzymes that charge specific tRNAs with their cognate amino acids and play an essential role in the initial steps of protein synthesis. Because these enzymes are attractive targets for drug development in many microorganisms, there is a pressing need for assays suitable for compound screening. We developed (1) a high-throughput assay for measuring aminoacyl-tRNA synthetase activity and (2) an accompanying method for preparing the tRNA substrate. The assay can be performed in 96-well plates and relies on malachite green detection of pyrophosphate (Pi) as an indicator of aminoacyl-tRNA synthetase activity. Analysis of Trypanosoma brucei isoleucyl-tRNA synthetase (IleRS) activity showed that the assay exhibits sensitivity to picomoles of product and yielded a Z' factor of 0.56. We show that this assay is applicable to other aminoacyl-tRNA synthetases and to enzyme inhibition studies. Using this assay, we found that the compound NSC616354 inhibits recombinant IleRS with an IC50 of 0.6 µM. Enzymology studies were also performed with rIleRS and its Km and kcat determined as 3.97 × 10(-5) mol/L and 312 S(-1), respectively. This assay will facilitate the screening of compounds to identify inhibitors of aminoacyl-tRNA synthetases.