In vitro systems that can effectively model liver function for long periods of time are fundamental tools for preclinical research. Nevertheless, the adoption of in vitro research tools at the earliest stages of drug development has been hampered by the lack of culture systems that offer the robustness, scalability, and flexibility necessary to meet industry's demands. Bioreactor-based technologies, such as stirred tank bioreactors, constitute a feasible approach to aggregate hepatic cells and maintain long-term three-dimensional cultures. These three-dimensional cultures sustain the polarity, differentiated phenotype, and metabolic performance of human hepatocytes. Culture in computer-controlled stirred tank bioreactors allows the maintenance of physiological conditions, such as pH, dissolved oxygen, and temperature, with minimal fluctuations. Moreover, by operating in perfusion mode, gradients of soluble factors and metabolic by-products can be established, aiming at resembling the in vivo microenvironment. This chapter provides a protocol for the aggregation and culture of hepatocyte spheroids in stirred tank bioreactors by applying perfusion mode for the long-term culture of human hepatocytes. This in vitro culture system is compatible with feeding high-throughput screening platforms for the assessment of drug elimination pathways, being a useful tool for toxicology research and drug development in the preclinical phase.