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Summary: Three-dimensional organoids, which resemble the pathophysiology and structural architecture of the original tissues, are a preferable in vitro model system for assessing metabolic activities in response to various environmental or nutritional changes. Here, we describe step-by-step protocols to establish and culture mouse and human hepatic tumor organoids. We also describe two straightforward and efficient approaches to harvest tumor organoids for investigating the effects of metabolites/drugs on viability and metabolic functions of tumor organoids.For complete details on the use and execution of this protocol, please refer to Tong et al. (2018), Leung et al. (2020), Tong et al. (2021), and Xu et al. (2021). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.