Proteolytic cleavage, either by viral or host proteases, is essential for maturation and full activity of many viral proteins. Consequently, antiviral development often targets viral encoded proteases. Our lab developed a cell-based assay to monitor viral protease activity, using HIV-1 protease (PR) as proof of principle. The assay relies on the inducible expression of PR fused between the DNA-binding and transactivation domains of the yeast transcription factor, Gal4. In the presence of an effective PR inhibitor, the fusion protein binds the Gal4 responsive element, and activates the green fluorescent protein (GFP) reporter. Dengue virus (DenV) is the causative agent of DenV Fever and Hemorrhagic Fever. There are no antivirals against DenV on the market, leaving bedside care as the only treatment available against one of the biggest threats to global public health. This project aims to adapt the HIV-1 PR assay (referred to as cytosolic assay) to the DenV PR non-structural protein 3 (NS3). DenV NS3 is a serine PR that requires the NS2B co-factor for proteolytic activity and proper folding. NS2B has four transmembrane domains (TM), two on either side of a hydrophilic cytoplasmic-exposed region. In order to monitor the activity of DenV PR in the context of the assay, we had to remove the NS2B TMs to allow the fusion protein to travel to the nucleus. Additionally, this project aims to develop a secondary assay to be used as a counter screen. This assay utilizes Gal4 and GFP in a novel manner, where rather than deleting the NS2B TMs, we exploit them to anchor our constructs in the Endoplasmic Reticulum membrane. It is worth noting that the virus naturally embeds its proteome in the Endoplasmic Reticulum membrane exploiting its many TMS, including those of NS2B. For this assay, referred to as the anchored assay, we engineered an intact Gal4 downstream NS2B/NS3 and a well-known NS3 cleavage site. Gal4 is released upon cleavage, thus activating GFP only when PR is active. This novel assay, in conjunction with the cytosolic assay, which has an opposite readout, will increase the potential of finding inhibitors when we embark in drug screening.