Flaviviral NS3 serine proteases require the NS2B cofactor region (cNS2B) to be active. Recent crystal structures of WNV (West Nile virus) protease in complex with inhibitors revealed that cNS2B participates in the formation of the protease active site. No crystal structures of ternary complexes are currently available for DENV (dengue virus) to validate the role of cNS2B in active site formation. In the present study, a GST (glutathione transferase) fusion protein of DENV-2 cNS2B49-95 was used as a bait to pull down DENV-2 protease domain (NS3pro). The affinity of NS3pro for cNS2B was strong (equilibrium-binding constant <200 nM) and the heterodimeric complex displayed a catalytic efficiency similar to that of single-chain DENV-2 cNS2B/NS3pro. Various truncations and mutations in the cNS2B sequence showed that conformational integrity of the entire 47 amino acids is critical for protease activity. Furthermore, DENV-2 NS3 protease can be pulled down and transactivated by cNS2B cofactors from DENV-1, -3, -4 and WNV, suggesting that mechanisms for activation are conserved across the flavivirus genus. To validate NS2B as a potential target in allosteric inhibitor development, a cNS2B-specific human monoclonal antibody (3F10) was utilized. 3F10 disrupted the interaction between cNS2B and NS3 in vitro and reduced DENV viral replication in HEK (human embryonic kidney)-293 cells. This provides proof-of-concept for developing assays to find inhibitors that block the interaction between NS2B and NS3 during viral translation.