The transfer RNA (tRNA)-dependent pathway for lipid aminoacylation is a two-step pathway composed of (1) a tRNA aminoacylation step catalyzed by an aminoacyl-tRNA synthetase, forming a specific aa-tRNA, and (2) a tRNA-dependent transfer step in which the amino acid acylating the tRNA is transferred to an acceptor lipid. The latter step is catalyzed by a transferase located within the cytoplasmic membrane of certain bacteria. Lipid aminoacylation modifies the biochemical properties of the membrane and enhances resistance of some pathogens to various classes of antimicrobial agents and components of the innate immune response. Lipid aminoacylation has also been linked to increased virulence of various pathogenic bacteria. Inhibition of this mechanism would render pathogens more susceptible to existing drugs or to natural defenses of a host organism. Because lipid aminoacylation is widespread in many bacterial genera and absent from eukaryotes, and because the tRNA aminoacylation step of this pathway is also used in protein biosynthesis (a process essential for bacterial life), this pathway represents an attractive target for drug design. We have reconstituted the lipid aminoacylation pathway in vitro and optimized it for high-throughput screening of libraries of compounds to simultaneously identify inhibitors targeting each step of the pathway in a single assay.