Many endemic, emerging, re-emerging or potentially emerging RNA viruses (Flavi-, Alpha-, Henipa-, Rhabdo-, Corona-viruses…) target the human central nervous system (CNS), causing severe neurological disorders, sometimes fatal or leading to debilitating consequences. The Flavivirus genus includes many of these highly neuropathogenic viruses (West Nile virus, Tick-borne encephalitis virus-TBEV, Zika virus…). Despite their dangerousness, only few vaccines exist and there is currently no available antiviral treatment. Whereas many efforts are made for the identification of such antiviral molecules, including with “broad-spectrum” properties, most studies use models that are not physiologically relevant. This may explain the lack of antiviral activity or excessive toxicity often observed in clinical trials, leading to their failure. Here, by comparing the antiviral activity of 8 selected molecules on 3 different models of infection of different relevance (A549, human neural progenitor cells and human neuronal/glial cells), we indeed demonstrated major differences in their capacity to inhibit viral infection, depending on the cellular model used : whereas most of the molecules had an antiviral activity in the A549 cell line, only one of them was efficient in TBEV-infected human neuronal/glial cells (hNGCs), the most physiologically relevant model. These results confirmed the importance of developing physiologically relevant models for testing or screening drugs for their antiviral activity. We thus aimed at developing an image-based phenotypic screen using the in vitro two-dimensional (2D) culture of neuronal/glial cells differentiated from fetal human neural progenitor cells, which we had previously shown that it reproduces major hallmarks of TBEV infection in the human brain. Using this unique screen, we identified new antiviral compounds. Two of them may be therapeutically repositioned in the future as they are drugs currently used in human therapy. Next, to further improve the physiological relevance of in vitro models, we developed a model of TBEV infection using 3D-cerebral organoids. We showed that one of the molecule previously identified exerts a strong antiviral activity in this model, confirming its potential interest for the treatment of human tick-borne encephalitis. We hope that, by using these physiologically relevant in vitro models of TBEV infection, we will select compounds with a very high probability to be efficient and non-toxic in the human brain.