Recent studies led to the identification of novel druggable targets in neuroblastoma (NB), opening the way for more effective treatments. However, children with high-risk NB still show high mortality rates, warranting efforts to develop novel therapeutic approaches. The repositioning of FDA-approved molecules is a strategy that has already been successfully employed to discover new applications for existing drugs. Therefore, we performed a high-throughput screening of a library of anti-cancer compounds (Selleckchem), consisting of 349 small molecules, either FDA-approved or under clinical trials, in order to repurpose drugs for NB therapy. The library was tested in three-dimensional multicultural spheroids, recognized as a reliable pre-clinical model to recapitulate drug responses in solid tumors. In the primary screening three NB cell lines (CHP134, IMR-32, SK-N-BE(2)) were treated for 72 hours with the library compounds at 10 μM concentration. Viability of 3D spheroids was evaluated using a high-content imaging approach. Images were processed using in-house developed algorithm in Harmony software (PerkinElmer). Finally, the data were analyzed employing the Strictly Standardized Mean Difference (SSMD) metric. We classified a compound as primary hit when it demonstrated SSMD 50 values for each of the 26 tested compounds were supplemented with available data on severe side-effects, therapeutic index, and molecular targets. In conclusion, our survey prioritized four candidate drugs, belonging to a class of multi-target tyrosine kinase inhibitors that potentially might be promising for repositioning in NB.