Alkhurma virus (ALKV) is a tick-borne class 4 flavivirus responsible for several human cases of haemorrhagic fever in Saudi Arabia, with no specific treatment currently available. The viral RNA encodes a serine protease (NS2B-NS3), essential for virus replication in infected cells, that constitutes an attractive target for antiviral compounds. In an attempt to identify residues and motifs on NS2B that are necessary for protease activity of the ALKV NS2B-NS3 complex, a series of modified NS2B-NS3 proteins was constructed, with point mutations on particular residues or with the NS2B domain derived from two different viruses. Four mutants and the two chimeric proteins exhibited reduction of protease activity against BAPNA (a p-nitroanilide substrate). The results demonstrate that tight complementarity of the protein sequences is necessary for NS2B-dependent activation of NS3. The results also determine residues in the ALKV NS2B cofactor essential for protease activation, giving new insights into protease function in flaviviruses.