In order to elucidate the localization of post-translational modifications (PTM) of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide and “S type” Cystatins) a comparative RP-HPLC-ESI-MS analysis on intact proteins of enriched-granule preparations from Parotid and Submandibular glands as well as Parotid, Submandibular/Sublingual (Sm/Sl) and whole saliva was performed. The main results of this study indicate that: i) phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage; ii) in agreement with previous studies, basic PRPs are secreted by the Parotid gland only, while all isoforms of acidic PRPs (aPRPs) are secreted by both Parotid and Sm/Sl glands; iii) phosphorylation levels of aPRPs, Histatin 1 and Statherin are higher in the Parotid gland, while the extent of cleavage of aPRP is higher in Sm/Sl glands; iv) O-sulfation of tyrosines of Histatin 1 is a PTM specific for the Submandibular gland; v) the concentration of Histatin 3, Histatin 5 and Histatin 6, but not Histatin 1, is higher in Parotid saliva; vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation and it occurs to the same relative extent in both glands; vii) the proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in Parotid secretion; viii) basic PRPs are cleaved in the oral cavity by unknown peptidases generating various small proline-rich peptides; ix) C-terminal removal from Statherin is more extensive in Parotid saliva; x) P-B peptide is secreted by both glands and its relative quantity is higher in Submandibular/Sublingual secretion; xi) in agreement with previous studies, “S type” Cystatins are mainly the product of Sm/Sl glands.