Changes in salivary composition correlate with disease susceptibility, disease state, or both. However, use of saliva for diagnostic purposes is complicated by the gland-specific effects of circadian rhythm or diurnal variation. We recently characterized a suite of peptides in the < or =10-kDa fraction of human parotid saliva that included many novel species. In this study, we used novel iTRAQ labeling chemistry to investigate possible diurnal effects on peptide generation. We collected samples produced by gustatory stimulation as the ductal secretions at four time points under conditions that minimized proteolysis, pooled them according to collection time, and isolated the LMW fractions. Samples collected at each collection time were derivatized with a different isobaric iTRAQ reagent. The labeled samples were combined, separated by reversed-phase HPLC, co-spotted with matrix on MALDI targets, and analyzed by MALDI TOF/TOF mass spectrometry. With this approach, we achieved relative quantification of the parotid peptides at four time points. In several cases, abundance during the day changed dramatically. iTRAQ tagging improved the efficiency of MS/MS fragmentation, which in turn allowed the identification of several novel peptides. Our results demonstrated both the utility of this method and the importance of diurnal effects on the composition of the human parotid saliva peptidome.