Organs-on-chip are increasingly catching on as a promising and valuable alternative to animal models, in line with the 3Rs initiative, to create 3D tissue microenvironments in which cells behave physiologically and pathologically at unparalleled precision and complexity. Indeed, these platforms offer new opportunities to model human diseases and test the potential therapeutic effect of different drugs as well as their limitations, overtaking the limited predictive accuracy of conventional 2D culture systems. Here, we present a liver-on-a-chip model to investigate the effects of two naturally occurring polyphenols, namely Quercetin and Hydroxytyrosol, on non-alcoholic fatty liver disease (NAFLD) using a method of high-content analysis. NAFLD is currently the most common form of chronic liver disease, whose complex pathogenesis is far from being clear. Besides, no definitive treatment has been established for NAFLD so far. In our experiments, we observed that both polyphenols seem to restrain the progression of the free fatty acid-induced hepatocellular steatosis, showing a cytoprotective effect due to their antioxidant properties. In conclusion, the resulting insights of the present work could guide novel strategies to contrast the onset and progression of NAFLD.