Background: The purpose of our study is to identify serum protein biomarkers for node-positive oral squamous cell carcinoma (OSCC). Biomarkers indicating lymph node metastasis provides valuable classification methodology to optimize treatment plans for patients with OSCC. Methods: Quantitative serum proteomic analysis of OSCCs with either node-positive or node-negative disease was performed with tandem mass spectrometry and isobaric tagging for relative and absolute quantitation (iTRAQ). Immunoassays were used to validate a panel of candidate protein biomarkers and receiver operating characteristic analysis was used to evaluate the performance of the candidate biomarkers. Results: A total of 282 serum proteins were quantified between node-positive and node-negative OSCCs with the proteomic approach. Four candidate biomarkers, gelsolin, fibronectin, angiotensinogen and haptoglobin, were validated in an independent group of patients with node-positive or node-negative OSCC. The best candidate biomarker, gelsolin, yielded a receiver operating characteristic value of 89 % for node-positive OSCC, although the sample size for validation is relatively small. Fibronectin, gelsolin and angiotensinogen were also found to be differentially expressed between cancer cell lines of node-positive and node-negative cancer origin. Conclusion: Our studies suggest that testing of serum protein biomarkers might help detect lymph node metastasis of oral cancer. Due to limited sample size in our studies, long-term longitudinal studies with large populations of individuals with oral cancer are needed to validate these potential biomarkers. Head Neck, 2014.