Zika virus (ZIKV) is a human mosquito-borne positive-sense RNA virus, belonging to the Flaviviridae family. World Health Organization (WHO) classified this virus as an Emergency in 2016 and currently identifies Zika as a priority disease. Although symptoms are generally mild, a risk of neurologic complications including Guillain Barré Syndrome is associated with the infection in adults, while infection during pregnancy is responsible for microcephaly and other congenital malformations. Since no vaccine or commercialized antiviral targeting this virus are available, scientific efforts are currently focusing into the development of treatments allowing to efficiently limit ZIKV spread. Prompted by this unmet medical need, we conducted a screen of 51,520 small chemical compounds using a high-content imaging cell-based assay, monitoring Zika virus replication within Huh-7.5 cells by combining DAPI staining of cellular nuclei together with immunostaining of the Zika virus envelope protein. 99 candidates were identified and validated as inhibiting ZIKV replication of at least 50% at a concentration of 10 µM. Subsequent dose-response studies were performed to evaluate the effects of each compound on both virus replication and cytotoxicity and compounds showing a strong dose-response inhibitory effect on replication with weak cell toxicity were then selected for follow-up studies. Two compounds sharing a common structure presented a particularly promising antiviral activity with a selectivity index, calculated as the ratio of 50 % inhibitory (IC50) and 50 % viability (CC50) concentrations, greater than 30. This common chemical scaffold showed to specifically inhibit ZIKV, displaying an antiviral activity against several strains of both African and Asian lineages but no effect on other Flaviviruses tested. Its antiviral activity was confirmed with similar efficacy in more relevant models for ZIKV infection, including human monocyte-derived dendritic cells (hMDDCs), human neural progenitor cells (hNPC) and the placenta-derived choriocarcinoma cell line JEG-3. Time of addition kinetics as well as specific entry and replication assays excluded an inhibitory role during ZIKV entry, highlighting an antiviral role during the RNA replication step. This observation, in addition to the appearance of resistant mutant viruses upon selection in the presence of the drug, strongly suggested a non-structural protein of the virus as a target of the compound. Current efforts are ongoing to identify the specific viral target of the compound and to get more insights about its mechanism of action. In addition, Pharmacokinetics (PK) and in vivo efficacy studies will be performed in the near future to evaluate the therapeutic potential of this compound. In summary, taking advantage of a cell-based large-scale high-content screening approach to identify small chemical compounds showing an antiviral activity against ZIKV, we identified a chemical scaffold specifically targeting this Flavivirus, inhibiting its RNA replication step.