Anthrax is a deadly infection caused by spores of the gram-positive bacterium Bacillus anthracis. Part of the bacteriums virulence is derived from the lethal toxin bipartite protein complex that disrupts MAPKK signaling pathways important for host defense against the infection. The crucial enzymatic member of lethal toxin is lethal factor, a zinc-dependent metalloprotease identified as a prime drug target for inhibitor development. Concerns about the use of anthrax as a bioweapon have created a need for effective antitoxin compounds to supplement the limitations of current antibiotic and antibody treatments. Our lab has developed a series of anthrax lethal factor inhibitors that contain a 2-(2-sulfonamidophenyl)benzimidazole motif capable of selectively binding to Zn2+ in the active site of lethal factor. These compounds were evaluated for biological activity against anthrax lethal factor with an in vitro fluorescence resonance energy transfer (FRET) assay. Initially, the FRET assay proved to be challenging and demonstrated a need for further optimization to yield consistent data. To address these challenges, a systematic protocol optimization was conducted to improve the reproducibility and accuracy of the data. Testing the optimized protocol with a positive control, PY-2S, has significantly improved data reproducibility, indicating that this updated protocol may be capable of reanalyzing our inhibitor library.