Zika virus is a mosquito-transmitted flavivirus that causes devastating fetal outcomes in the context of maternal infection during pregnancy. An important target for drugs combatting Zika virus pathogenicity is NS2B-NS3 protease, which plays an essential role in hydrolysis and maturation of the flavivirus polyprotein. We identify hydroxychloroquine, a drug that already has approved uses in pregnancy, as a possible inhibitor of NS2B-NS3 protease by using a Food and Drug Administration-approved drug library, molecular docking, and molecular dynamics simulations. Further, to gain insight into its inhibitory potential toward NS2B-NS3 protease, we performed enzyme kinetic studies, which revealed that hydroxychloroquine inhibits protease activity with an inhibition constant (K i) of 92.34 ± 11.91 μM. Additionally, hydroxychloroquine significantly decreases Zika virus infection in placental cells.