Most viruses rely on the processing of their proteome by viral and/or host proteases (PRs). This is the case of Dengue Virus (DenV), a member of the Flaviviridae family and the most common and widespread arthropod-borne viral infection of humans. DenV currently has no effective vaccines or antiviral drug treatments. Developing antiviral drugs that target DenV NS2B cofactor/NS3 protease could potentially be one of the most successful strategies in combating DenV pathogenesis and spread. The primary goal of this project is to develop an assay that will provide a robust, cell-based, high throughput method to quickly identify novel PR inhibitors for DenV. Hilton and Wolkowicz developed a cell-based assay to monitor the catalytic activity of HIV-1 PR in T-cells. This project will adapt the assay from HIV-1 PR to the NS2B/NS3 protease complex of the four serotypes of DenV and monitor the proteolytic activity via a similar GFP reporter system. In addition, all of the necessary components of the reporter system and the DenV adapted Gal4/NS2B/NS3 fusion will be stably expressed in a genetically bar coded non-adherent T-cell line and an adherent hepatocyte cell line. The final version of the assay will be a cell-based, high throughput, multiplexed system designed to efficiently screen hundreds of thousands of candidate inhibitors of DenV NS3 protease activity. In addition, the assay can be utilized to further illuminate the cellular localization of various NS2B transmembrane deletion mutants, how these mutants effect NS3 protease activity ex vivo, and possibly discover novel cleavage targets of NS2B/NS3.