In the present study various proteoforms of cystatin A, cystatin B, cystatin D, cystatin S, cystatin SN and cystatin SA were detected and characterized by a top-down HPLC-ESI-MS platform integrated by bottom-up strategies in the acidic soluble fraction of human saliva. The several proteoforms derive from polymorphisms of the coding sequence and post-translational modifications, typically phosphorylation, N-terminal processing, and oxidation. The protein species detected in saliva collected from healthy subjects were: (i) cystatin AT96→M and its acetylated derivative; (ii) cystatin B N-terminally acetylated and carboxymethylated at C3; (iii) N-terminally truncated cystatin D with the N-terminal Q converted to pyro-E and lacking the first 5 amino acid residues (pGlu-cystatin D Des1-5); (iv) N-terminally truncated forms of cystatin SN and SNP11→L lacking the first 4 amino acids (cystatin SN Des1-4 and cystatin SNP11→L Des1-4) and the first 7 amino acids (cystatin SN Des1-7 and cystatin SNP11→L Des1-7); (v) N-terminally truncated cystatin SA lacking the first 7 amino acids (cystatin SA Des1-7); (vi) oxidized derivatives of cystatins SN and S1 at W23 and W107.