Summary Background: Mutational clonality arises when somatic mutations confer a growth advantage, leading to clonal expansion of genetically identical cells. These clones often harbor cancer-associated driver mutations and have been identified in normal tissues such as colon, oesophagus and bronchus, but remain unexplored in the head and neck region. Mapping this clonal architecture is crucial for understanding the earliest stages of head and neck squamous cell carcinoma (HNSCC) development. This work, part of a larger mutational clonality project, aimed to optimize epithelial layer isolation from post-mortem head and neck tissues to support downstream genomic analyses and to evaluate the presence of TP53 mutations in normal tissues using immunohistochemistry (IHC). Methods: Tissues from multiple head and neck sites were obtained from whole-body donors. Epithelial layers were enzymatically separated with Dispase II under varying temperatures. Isolation success was assessed by hematoxylin and eosin (H&E) staining. TP53 mutation status was probed via IHC using the PAb240 antibody specific for mutant p53 protein. Results: Isolation efficiency varied by temperature conditions, with 37 °C yielding the highest success. Oropharyngeal, laryngeal and skin samples were most efficiently isolated; buccal mucosa, floor of mouth and tongue were more challenging. Increased Dispase II concentration (2 mg/mL) improved separation in buccal mucosa and floor of mouth. H&E is confirmed to be valuable for identifying epithelial origin. IHC failed to detect mutant p53 protein in normal tissues despite optimization attempts. Conclusion: A reproducible isolation protocol was established, with H&E as a key validation tool. Further troubleshooting of p53 staining with the PAb240 antibody is needed to improve the success of IHC experiments. An optimized IHC protocol, combined with sequencing-validated mutant p53 tissues, could enable spatial-genomic mapping of clonal architecture in normal head and neck tissues.