The present pilot study aims to investigate the diagnostic and prognostic efficacy of serum HSP90 beta in Head and Neck Squamous Cell Carcinoma (HNSCC) patients subjected to localized hyperthermia therapy (HT). Serum levels of HSP90 beta were measured by ELISA and its diagnostic and prognostic efficacy was determined by receiver operating characteristic curve (ROC) analysis. HNSCC patients showed significantly (P<0.05) higher serum levels of HSP90 beta (65.6±13.08 ng/ml) compared to Healthy Controls (HC: 23.5±3.8 ng/ml). No significant difference was observed in serum HSP90 beta levels between complete responders (CR) and non-responders (NR) in the chemo-radiation therapy (CRT) cohort. However, in CRT+HT cohort, CR showed significantly (P = 0.02) lower serum HSP90 beta levels at 24 h after HT (25.6±9.04 ng/ml) compared to NR (130.5±34.2 ng/ml). Youden’s index values between HNSCC versus HC, CR versus NR (CRT) and CR versus NR (CRT+HT) were found to be 0.47, 0.45 and 0.80, respectively. Thus, alterations in the serum HSP90 beta after HT suggest its potential in prognosis of HT response in HNSCC patients. Elevated levels of HSP90 beta may serve as a promising diagnostic serum bio-marker for HNSCC. However, further validation in larger patient samples is needed for clinical translation of HSP90 beta as diagnostic and prognostic biomarker.