The in vitro models to study of Hepatitis B virus (HBV) are very limited and only poorly mimic physiological virus-host interaction. Recently, we have demonstrated that 3D microfluidic cultures are a powerful tool to study HBV within its natural target cell. These cultures allow the long-term survival of primary human hepatocyte, in the presence and in the absence of Kupffer cells, are highly susceptible to HBV and recapitulate all steps of the HBV life cycle. In addition to mimicking host responses observed in HBV-infected patients, these advantages enable the evaluation of sequential and long-term antiviral treatments targeting any step of the viral cycle and the study of innate immune responses to HBV by hepatocytes and other non-parenchymal cells in the liver.