Dengue Virus is a mosquito-borne arbovirus within the family Flaviviridae. An estimated 390 million cases of infection occur annually, however currently neither prophylactic nor therapeutic options exist [1]. An attractive target for antiviral drugs is the virally encoded serine protease (NS3pro) and its associated cofactor (NS2B). The NS2B-NS3pro complex is required for cleaving the viral polyprotein into the 10 separate proteins, and is therefore essential for replication and infectivity [2]. Current attention is focused on developing small-molecule inhibitors which target the protease active-site, however the development of effective inhibitors has posed significant challenges. An inhibitor approach based on NS2B cofactor displacement could prove to be an effective alternative. For the majority of past research, the NS2B-NS3pro complex has been expressed through linking the C-terminus of NS2B to the N-terminus of NS3pro via a flexible glycine linker [3]. In this research, the protease was expressed as a catalytically active, unlinked NS2B-NS3pro complex. Theoretically, the non-covalent association should allow a more native context for examining inhibitors that target cofactor-protease interactions, as opposed to active-site inhibitors. Site-directed alanine mutagenesis was conducted on residues 75-87 of NS2B, in addition to 11 residues of NS3pro that intimately engage with this site. Activity was assayed via measuring cleavage of a chromogenic substrate over time [3, 4]. Alanine mutagenesis of 10 residues resulted in a reduction of proteolytic activity to <20%. Mapping of these residues on the pre-existing crystal structure [5, 6] showed 6 of these residues to be located together in a hydrophobic pocket. Interestingly, this hydrophobic region is only apparent in the ligand-bound conformation of the complex. In comparison, when NS2B-NS3pro is ligand-free, these residues do not interact. It is possible that mutation of these residues impedes the conformational change which allows ligand binding. The findings have provided mechanistic insights to the functional role the cofactor may play in substrate binding. The hydrophobic site identified may provide a novel target site for inhibitor development.