Oral squamous cell carcinoma (OSCC) is the fifth most common cancer in Taiwan and the fourth in males. Oral cancer is associated with high death rate mainly because >1/2 patients were diagnosed at advanced stages that lead to poor prognosis. At present, there are still no suitable biomarkers for detecting early-stage oral cancer. Aberrant glycosylation is known highly associated with cancers. Saliva is an easy-to-obtain body fluid derived from oral cavity and directly contacts oral cancer nidus, representing an excellent resource for identifying novel, noninvasive biomarkers for detecting OSCC. Here, we systemically compared the difference of glycoproteins in saliva samples from healthy controls (HC), subjects with oral potentially malignant disorders (OPMD) and OSCC patients using lectin-based glycoprotein/glycopeptide enrichment coupled with iTRAQ-based quantitative approach. From this analysis, 673 proteins (353 glycoproteins) containing 2366 peptides (834 glycopeptides) were identified and quantified; among them, 15 glycopeptides (derived from 14 proteins) showed significantly higher expression in OSCC saliva and were selected as targets for further verification. We then established multiple reaction monitoring (MRM)-MS assays for these 15 glycopeptides and quantified their levels in 90 individual saliva samples (30 HC, 30 OPMD and 30 OSCC). The results showed that eight glycopeptide targets displayed significantly elevated levels in OSCC compared with HC or OPMD groups. Collectively, we have identified several novel biomarkers from saliva via glycoproteomic and MRM-MS analysis which may have potential to be further validated for their clinical use in oral cancer detection.