The objective of this study was to assess the effect of pre-analytical processing on proteomic analysis of saliva and to identify salivary biomarkers for potential clinical applications. Saliva samples from five healthy individuals and three head and neck squamous carcinoma (HNSC) patients were initially depleted of major protein constituents. Saliva from healthy subjects was divided and processed by three different methods prior to liquid chromatography and tandem mass spectrometry technique (LC-MS/MS) analysis. The results showed marked differences amongst the methods. The SDS-PAGE separation and in-gel digestion method yielded the highest number of proteins that included the majority of those identified by the other two methods. The in gel-digestion method was used in the LC-MS/MS analysis of saliva from three HNSC patients and the results were compared with those from healthy subjects. Our analysis identified two proteins, alpha-1-B-glycoprotein and complement factor B proteins, to be present in patients but not in normal specimens. Paradoxically, cystatin S, parotid secretory factor, and poly-4-hydrolase beta-subunit proteins were detected in most normal salivas but not in patient specimens. Subsequent analysis of complement factor B by Western blotting showed strong immunoreactive bands of complement factor B in HNSC patients' and negative or weakly positive in normal saliva samples. We conclude that 1) initial saliva processing affects protein analysis, 2) in-gel digestion followed by LC-MS/MS detects the most saliva proteins, 3) certain proteins are differentially found in patient and normal salivas and 4) a small set of proteins can be targeted for future validation for clinical investigation.