We describe a proof-of-concept study of a competitive enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) with active-site-directed probes coupled to a 5-O-N-(aminoacyl)sulfamoyladenosine scaffold. A biotin functionality immobilizes the probes onto a streptavidin-coated solid support. Dissociation constants were determined with a series of ligands, including enzyme substrates and a library of sulfamoyloxy-linked aminoacyl/aryl-AMP analogues. As it enables direct readout of protein-ligand interaction, the competitive ELISA technique provided information on comparative structure- activity relationships and insights into the enzyme active-site architecture of NRPS A-domains. These studies indicate that the ELISA technique can accelerate the discovery of small-molecule inhibitors of the A-domains with new scaffolds that perturb the production of NRPS-related virulence factors.