Head and neck squamous cell carcinoma (HNSCC) is a diverse cancer with high molecular and clinical heterogeneity. HNSCC is the sixth-most common cancer worldwide, with 890,000 new cases and 450,000 deaths. Despite the intense work of scientists to advance the field of medicine, HNSCC patients' overall survival (OS) remains below 50%. This is mostly because of treatments failure, poor prognosis, and a limited number of pre-clinical models that can be used to study and show how HNSCC works. Our study aimed to use a hamster PDX model for HNSCC by creating spheroids/organoids (S/Os) from two HNSCC cell lines (CAL-27 and FaDu) and then classify them at the proteome level. We also cultivated cancer-associated fibroblasts (CAFs) in 2D cultures to prepare them for transplantation alongside the S/Os. Finally, we used histology and IHC to see how well these S/Os and CAF cells work in the xenotransplantation system. Our findings show that incorporating CAFs with HNSCC S/Os has a substantial effect on transplant mass growth and progression in the PDX model. We also assessed the expression of specific proteins (AR, p-FAK, CD133, CD44, CD8α, NFkB, PCNA, P53, Ki-67, HOX11, TR4, and YY1) in the donor S/Os and transplant masses. The results indicate that CAFs can impact particular cellular processes, including proliferation, adhesion, and immune response and the marker proteins’ differential expression and location reveal potential therapeutic targets and provide insight into the processes by which CAFs may advance HNSCC. Our findings conclude that incorporating CAFs with HNSCC S/Os influences tumor growth and progression in the hamster xenotransplantation model and that this model shows effectiveness for the preclinical studies of HNSCC.