The dengue virus protease NS3pro/NS2B, a key antiviral target for drug development, has been previously reported to adopt either an “open” or a “closed” conformation in an ensemble of crystal structures with different NS2B C-terminus (NS2B) positioning. In this study, in order to unambiguous identify the specific conformational ensembles that dominates in solution we apply a novel strategy, referred to as conformational filter, based on Nuclei Magnetic Resonance (NMR) spectroscopy complemented by Molecular Dynamic (MD) calculations. This was achieved by comparison of the experimental values of the relaxation parameters in the fast dynamic time window of the back bone and methyl side chains with corresponding back calculated relaxation parameters of the different conformational ensembles obtained from free MD simulations. Our analyses of the relaxation data averaged over the ensembles populated indicate that “open” conformation of DENV-2 NS3pro/NS2B registered by X-ray but not in solution is absent or below the detection threshold. Based on this data we claim that the “open” conformation found for this part of the crystal structure of NS3pro/NS2B is probably due to crystal contacts. Importantly that conformational ensembles selected through NOE restrained MD as well as observed in crystallisation where position of co factor NS2B is located in so called “partly” open conformation was in full agreement with the conformational filter: experimental and free MD calculated relaxation parameters showed good agreement. Additionally we unambiguously showed a high probability for the conformational ensemble of the “closed” conformation.