Arbovirus infections became epidemics in recent decades due to globalization, urbanization, and climate change. Dengue Virus (DenV), belonging to Flaviviridae, is one of the most prevalent mosquito-transmitted viruses leading to Dengue Fever. No antiviral treatments exist currently. As part of the mechanisms of infection, the flavivirus protease cleaves both viral and host proteins. The nonstructural protein 3 (NS3) is a serine protease that utilizes the NS2B cofactor for enhanced proteolytic activity. Previously we used a bioinformatics approach based on viral cleavage-site sequences to search for potential substrates within the human proteome. Hence, we have created a cellular-based assay aimed at deciphering the function and properties nature of human proteins as true substrates of the DenV NS2B/NS3 proteases. The in trans DenV PR cellular assay utilizes the DNA binding and the transactivating domains of the Gal4 yeast transcription factor. When fused, the domains bind to the Gal4 promoter, which drives green fluorescence protein (GFP) expression. Prior experiments with HIV and DenV proteases were performed with the viral protease fused within the Gal4 domains and monitored protease activity in cis. In these assays, fluorescence was observed only with inactive protease. Here, I have explored utilizing the Gal4 PR assay for in trans cleavage. For that purpose, NS3/NS2B is supplied in trans, and the putative substrates cleavage sites replace protease within the Gal4 domains. We tagged the Gal4 domains with FLAG and HA epitopes to allow for the detection of cleavage by Western blotting. As proof of principle and positive control, we have used twenty amino acids of the well-characterized viral NS4B/NS5 cleavage site. Flow cytometry and Western blotting were used to confirm cleavage. We have explored numerous forms of NS2B/NS3 that have shown cleavage in both cellular assays and protein extract experiments. These different forms of protease did not show cleavage. Future experiments may explore possible steric hindrances of either the DNA binding domain or transactivating domain of Gal4 and the substrate. Although G2S linkers were placed upstream and downstream of the substrate cleavage site, the two domains of Gal4 that obscures the substrate and prevents the cleavage event.