Dengue virus remains a public health threat. Dengue NS2B-NS3 proteins are prime antiviral drug targets and have been studied extensively. Here, we combine cross-linking mass spectrometry, molecular dynamics simulations and biochemical assays to identify NS2B-NS3 full length interactions. Using cross-linkers of different lengths as molecular rulers, we identified NS2B S48 as a key interacting residue that has not been previously reported. Molecular dynamics simulations modelled a compact conformation of NS2B to NS3 interaction with better agreement to cross-link data. In contrast, previously reported crystal structure indicated an elongated conformation with poor agreement to cross-link data. Mutation of NS2B S48 to alanine or lysine greatly reduces protease activity and disrupts the NS2B S48 binding pocket in the compact conformation. NS2B-NS3 cross-links were also found to be conserved across all four dengue serotypes. Our interdisciplinary approach reveals new key interacting residue and a compact conformation that can guide drug development against dengue.