LAUSR

&NA; Zika virus (ZIKV) infection may be associated with severe complications in fetuses and adults, but treatment options are limited. We performed an in silico structure‐based screening of a large chemical library to identify potential ZIKV NS2B‐NS3 protease inhibitors. Clinically approved drugs belonging to different drug classes were selected among the 100 primary hit compounds with the highest predicted binding affinities to ZIKV NS2B‐NS3‐protease for validation studies. ZIKV NS2B‐NS3 protease inhibitory activity was validated in most of the selected drugs and in vitro anti‐ZIKV activity was identified in two of them (novobiocin and lopinavir‐ritonavir). Molecular docking and molecular dynamics simulations predicted that novobiocin bound to ZIKV NS2B‐NS3‐protease with high stability. Dexamethasone‐immunosuppressed mice with disseminated ZIKV infection and novobiocin treatment had significantly (P < 0.05) higher survival rate (100% vs 0%), lower mean blood and tissue viral loads, and less severe histopathological changes than untreated controls. This structure‐based drug discovery platform should facilitate the identification of additional enzyme inhibitors of ZIKV.