Alkhumra virus, a zoonotic pathogen in the Flaviviridae family, causes severe hemorrhagic fever in humans, yet vaccines and drugs remain unavailable. The NS2B/NS3 protease, essential for virion maturation, represents a promising therapeutic target. Structural and dynamical changes induced by NS2B cofactor binding to the NS3 protein were examined using all-atom molecular dynamics simulations. NS2B binding reduces the flexibility of NS3, particularly in contact regions, without altering its secondary structure. Non- bonding van der Waals and electrostatic interactions are identified as the primary driving forces in cofactor binding. The protonation states of catalytic triad residues significantly affect the active pocket’s geometry. A drug repurposing campaign utilizing ensemble docking and molecular dynamics simulations identified three DrugBank compounds as potential NS2B/NS3 protease inhibitors. The catalytic serine residue with a deprotonated hydroxyl group contributed most significantly to the free energy of binding. These findings provide a detailed understanding of the molecular interactions underlying ligand binding to NS2B/NS3, offering valuable insights for developing effective inhibitors.