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Summary: The lack of suitable models currently hampers our understanding of how the tumor microenvironment responds to immunotherapy treatment. Here, we present a protocol for ex vivo culture of patient-derived tumor fragments (PDTFs). We describe the steps for tumor collection, generation and cryopreservation of PDTFs, and their subsequent thawing. We detail culture of PDTFs and their preparation for analysis. This protocol preserves the tumor microenvironment’s composition, architecture, and cellular interactions, which can be perturbed by ex vivo treatment.For complete details on the use and execution of this protocol, please refer to Voabil et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.