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Summary: Tumors originating from the head and neck represent diverse histologies and are comprised of several cell types, including malignant cells, cancer-associated fibroblasts, endothelial cells, and immune cells. In this protocol, we describe a step-by-step approach for the dissociation of fresh human head and neck tumor specimens, followed by isolation of viable single cells using fluorescence-activated cell sorting. Our protocol facilitates the effective downstream use of techniques, including single-cell RNA sequencing and generation of three-dimensional patient-derived organoids.For complete details on the use and execution of this protocol, please refer to Puram et al. (2017)1 and Parikh et al. (2022).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.