Drug hepatotoxicity testing requires in vitro hepatocyte culture to maintain the long-term and stable liver specific functions. We developed a drug testing platform based on laminar-flow immediate-overlay hepatocyte sandwich perfusion culture. The immediate-overlay sandwich (collagen-coated porous polymeric membrane as top overlay) protects the cells and integrity of the top collagen matrix from the impact of flow. A bioreactor was designed that allowed proper control of shear stress and mass transfer. The culture parameters such as the optimal perfusion initiation time and flow rate were systematically and mechanistically determined. The optimized system could re-establish hepatocyte polarity to support biliary excretion and to maintain other liver specific functions, such as the biotransformation enzyme activities, for two weeks that extended the usable in vitro hepatocyte-based drug testing window. When the perfusion cultured hepatocytes from days 7 or 14 were used for drug testing, the APAP-induced hepatotoxicity measurements were more sensitive and consistent over time than the static culture control, enabling further exploitations in large-scale drug testing applications.