The pathogenic West Nile virus (WNV) and Dengue virus (DV) are growing global threats for which there are no specific treatments. Both viruses possess a two component NS2B/NS3 protease which cleaves viral precursor proteins. Whereas for the WNV protease two crystal structures in complex with an inhibitor have been solved recently, no such information is available for the DV protease. Here, we report the generation of a homology model of DV NS2B/NS3 protease. Since it is known from the related WNV protease that it adopts a distinct conformation in free and in inhibitor-complexed form, a special emphasis was given to the analysis of the protease flexibility. Therefore, several models of DV NS2B/NS3 protease complexed with the peptidic inhibitor (Bz-Nle(P4)-Lys(P3)-Arg(P2)-Arg(P1)-H) were generated. The first DV protease model (DV-1) was constructed using the available crystal structure of the apo DV NS2B/NS3 protease. The second model (DV-2) was built taking the WNV NS3/NS2B protease in the inhibitor-complexed form as the template structure. Molecular dynamics simulations which were carried out for the WNV crystal structures as well as for the DV models provided an understanding of the role of NS2B for maintaining the protease in the active conformation. It was also demonstrated that NS2B is not only important for maintaining NS3 in the active form, but is also essential for establishing the interaction between residues from the S2 pocket and the peptidic inhibitor. The DV NS2B/NS3 model in the productive conformation can now be used for structure-based design purposes. Copyright © 2009 John Wiley & Sons, Ltd.