Oral squamous cell carcinoma (OSCC) is the fifth most common tumor causing high mortality in Taiwan, but currently there are still no available biomarkers for detecting early-stage OSCC. Abnormal glycosylation is known as a pivotal regulatory mechanism in tumor malignancy, and saliva is a non-invasive body fluid derived from oral cavity with abundant glycoproteins. Recently, we have applied iTRAQ-based quantitative glycoproteomics coupled with pisum sativum agglutinin (PSA) enrichment of glycopeptides to analyze the differential glycoproteome profiles in saliva samples from healthy controls, subjects with oral potentially malignant disorders and OSCC, from which several glycopeptides showing significantly increased levels in OSCC saliva were discovered. To facilitate the future verification of these target glycopeptides in saliva samples, the present study aims to develop an automatic KingFisher-assisted glycopeptides enrichment procedure. We chose toluenesulfonyl (tosyl)-activated magnetic beads to couple PSA and optimized the conditions for coupling maximal amounts of PSA to the beads and for blocking and pre-cleaning beads to reduce nonspecific binding of protein contaminants prior to LC-MRM-MS analysis. We also evaluated the appropriate amount of saliva sample for LC-MRM-MS assay using the afore-mentioned optimized procedure for glycopeptie enrichment. Afterward, we incorporated the optimized condition for the magnetic bead-based glycopeptides enrichment procedure into the KingFisher magnetic bead processor and successfully created a workflow for the entire process. We found that the KF-Flex-96-DW (deep well)-head magnetic base performed better than the KF-Flex-96-PCR-head magnetic base in fully suspending the magnetic beads (5 mm) and moving beads to other plates. This established workflow for automatic KingFisher-assisted glycopeptides enrichment procedure will be applied to quantify target glycopeptides in individual saliva samples from OSCC patients and non-OSCC subjects.