Head and neck squamous cell carcinoma (HNSCC) is the sixth most common neoplasm in the world. The follow-up protocols currently available do not appear to diagnose treatment failures and recurrences early enough to provide the best treatment to improve the survival rates of patients. The identification of biomarkers may aid in diagnosing, monitoring the progression, or predicting treatment outcomes in HNSCC. The present study aimed to evaluate whether cluster of differentiation (CD) 3ζ chain expression may serve as a biomarker for the early detection of recurrent or persistent HNSCC. However, in a longitudinal study, a standardized method that allows consistent data comparisons in an inter-assay manner is critical. The present study reveals a method to monitor expression levels of CD3ζ over multiple time-points using flow cytometry. The present study validated the use of an internal control and normalization procedure for tracking alterations in CD3ζ expression in samples from patients with HNSCC, which were collected and assayed for a longitudinal study.