Multidimensional liquid chromatography – mass spectrometry (LC-MS/MS) has been used for the analysis of biological samples labeled with isobaric mass tags (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous-cell carcinomas(HNSCCs) in relation to non-cancerous head-and-neck tissues (control) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six non-cancerous tissues) in five sets by online and offline separation. We identified 811 non-redundant proteins in HNSCCs, including structural proteins, signaling components, enzymes, receptors, transcription factors and chaperones. A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3 sigma), YWHAZ (14-3-3 zeta), three calcium-binding proteins of the S100 family, S100A 2, S100A 7 (psoriasin) and S100A 11 (calgizarrin), prothymosin alpha (PTHA), L-lactate dehydrogenase A chain (LDH-A), glutathione S transferase-Pi, APC-binding protein EB1, and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3 and polybromo-1D (BAF180) were underexpressed in HNSCCs. A panel of the three best-performing biomarkers – YWHAZ, stratifin and S100A7 - achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues. Verification of differential expression of YWHAZ, stratifin and S100A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry, immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin and S100A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous 3 head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers.