Hepatocytes, the cells responsible for the metabolic and detoxification processes in the liver, are the predominant target of hepatitis B virus (HBV) infections, a major cause of liver cancer. The limited availability of normal human hepatocytes for cell-culture based studies is a significant challenge in HBV-associated liver cancer research. Therefore, there is a need for miniaturized cell-culture systems that can serve as a platform for studying the effect of HBV infections on hepatocyte physiology. Here, we present a microfluidic platform that can be used to study HBV replication in both rat and human hepatocytes. Polydimethylsiloxane (PDMS) microchannels fabricated using soft lithography techniques served as a culture vessel for both primary rat hepatocytes (PRH) and a human hepatoblastoma cell line, HepG2. The micro cell-culture chamber was then used as a model for HBV replication studies. Cells were grown in static culture conditions and either transfected with an HBV-genome cDNA or infected with the viral genome expressed from a recombinant adenovirus. Supernatants collected from the microchannels were assayed for secreted HBV using polymerase chain reaction (PCR). We achieved approximately 40 and 10% transfection efficiencies in HepG2 cells and PRH respectively, and 80-100% adenoviral infection efficiency in PRH comparable to standard tissue culture methods. Moreover, we successfully detected replicated HBV using our novel platform. This platform can be easily extended to studies involving DNA transfection or HBV infection of primary human hepatocytes since only a small number of cells are required for studies in microfluidic chambers.