This work demonstrates that a comprehensive strategy of proteomic identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomic approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified, with 8 of these being reported for the first time in OSCC. Of the 8 newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia, as well as clinical stage, lymph node involvement and recurrence, these being known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, being not only closely related to cell proliferation and apoptosis, but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, monitoring in the therapy of oral squamous carcinoma. Further, this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.