Saliva has become one of the more attractive body fluids for protein biomarker discovery studies because of its ease of collection, non-invasiveness and multiple samples can be collected from an individual at a single time point. With the development of modern data acquisition strategies, such as Sequential Window Acquisition of all Theoretical Mass Spectrometry (SWATH-MS), the quality of saliva sample preparation has become a crucial factor for a successful protein identification and quantification. Several sample preparation methods have been proposed, but there has been no systematic evaluation conducted to date that compared each of these methods. We have therefore, performed an extensive assessment using technical and biological repeats to evaluate the number of protein IDs and repeatability of three most commonly used techniques, in-solution digestion, filter-aided sample preparation (FASP) and in stage-tip (iST) digestion. We discovered that in the case of human saliva sample, FASP provided the highest number of proteins (human and microbial) identifiable from a pool saliva sample, and there were no significant differences in terms of repeatability among the three methods investigated.