The liver plays an important role in a broad spectrum of physiological functions and detoxifies endogenous and exogenous substances. The liver failure is associated with a high risk of mortality because it is one of important organs in our body. Various bioartificial liver (BAL) systems have been used for clinical trials as a bridge for liver transplantations in patients with liver failure. Long term and stable liver-specific functions of hepatocytes in the development of BAL support systems should be considered. Spheroid formation of hepatocytes enhances liver-specific functions. In this review, hepatocyte spheroid formation methods such as galactose density, topology of extracellular matrix, micromolding technique, hanging-drop culture, non-adhesive surface, positive charged surface, spinner culture, rocked technique, medium component, external forces, coculture system and polymeric nanospheres are explained for enhancing liver-specific functions.