The growing number of bacterial pathogens developing resistance to all current antibacterial drugs is of great concern, as it leaves limited treatment options available for patients. However, very few new anti-bacterial compounds are being developed to treat bacterial infections. Understanding the mechanisms through which bacterial virulence is mediated, can help the development of novel antibacterial compounds. One of the pathways required for production of virulence factors in bacteria is the DiSulfide Bond (DSB) oxidative pathway. Disulfide bonds are essential for structural stability and functional ability of proteins. The most widely studied DSB pathway is in E. coli K-12, in which the proteins DsbA and DsbB act in concert to introduce disulfide bonds between two cysteine residues via an oxidative protein folding step. Here, we describe an assay where potential small molecule inhibitors of the soluble protein DsbA and the membrane protein DsbB can be screened. A time-resolved fluorescence assay has been used as a screening platform for potential inhibitors of the E. coli proteins DsbA and DsbB. Using 384-well plates to minimise reagents, compounds can be screened in a high-throughput format. The assay can also be used to generate dose-response curves of promising lead compounds for IC50 data analysis and comparison of potencies.