Head and Neck Squamous Cell Carcinoma (HNSCC) ranks sixth among the major causes of fatalities worldwide. With greater than 600,000 incidences reported annually, cancer scientists need to devise more effective therapeutic and diagnostic approaches to improve the life quality of HNSCC patients. Despite advancements in the field of medical science resulting in multimodal therapeutic strategies, the overall survival (OS) of HNSCC patients remains less than 50%. This is mainly due to chemotherapeutic resistance and a scarcity of clinically prognostic pre-clinical experimental models to reflect the pathobiology of head and neck tumors. Patient-Derived Xenografts (PDXs) are potential pre-clinical model systems for oncological research. PDXs are postulated to more closely mimic human tumor tissues compared to cultured cell lines in terms of histopathology, morphology, drug-resistance and/or chemosensitivity, recurrence, progression, and metastasis. We proposed to exploit the xenotransplantation strategy for HNSCC by developing and using three-dimensional culture systems (from two different HNSCC cell lines, CAL-27 and FaDu) to generate spheroid/organoid (S/O) cell populations. We characterized those donor cell populations at the proteomic level by Western blot and immunohistochemical analyses and then we generated and histologically assessed CAL-27 and FaDu cell S/O transplants in the hamster cheek pouch.