Hepatocytes in sandwich configuration constitute of primary hepatocytes cultured between two layers of extracellular matrix. Sandwich-cultured hepatocytes maintain expression of liver-specific proteins and gradually form intact bile canaliculi with functional biliary excretion of endogenous compounds and xenobiotics. Both freshly isolated and cryopreserved hepatocytes can be used to establish sandwich cultures. Therefore, this preclinical model has become an invaluable in vitro tool to evaluate hepatobiliary drug transport, metabolism, hepatotoxicity, and drug interactions. In this chapter, commonly used procedures to cultivate primary hepatocytes from human and rat in sandwich configuration are described.