OBJECTIVE To analyze surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein profiles of patients with head and neck squamous cell carcinoma (HNSCC) and healthy controls and to determine the sensitivity and specificity of SELDI assay for HNSCC detection before and after treatment. METHODS Proteomic analysis and comparison of serum samples. METHODS Tertiary care academic medical center. METHODS Seventy-eight patients with HNSCC and 68 healthy controls. METHODS Serum samples were prospectively collected from 78 patients with HNSCC and 68 healthy control volunteers. SELDI-TOF-MS was performed on serum samples to identify protein peaks in the range of 0 to 100 kDa. Classification analysis of the spectral data was performed and used to classify the disease status of the patients. RESULTS The SELDI-TOF-MS assay generated serum protein profiles ranging from 0 to 100 kDa. After background subtraction, mass calibration, and normalization, 545 protein peaks were identified. Classification tree analysis based on peak expression correctly classified patients with HNSCC with 82% sensitivity and 76% specificity. Subgroup analysis correctly classified 83% of oral cavity tumors, 81% of oropharyngeal tumors, and 88% of laryngeal tumors. Pretreatment and posttreatment samples were available from 12 patients, and the posttreatment samples were correctly classified in 86% of the patients at 3 months and 75% of the patients at 6 months. CONCLUSIONS Proteomic SELDI-TOF-MS analysis of serum protein profiles distinguishes patients with HNSCC from controls with a high degree of sensitivity and specificity. Further investigation into the clinical utility of this technology in HNSCC detection and surveillance is warranted.