Selective cleavage of the peptide backbone is one of the major obstacles in protein analysis. Peptide bond is stabilized by a resonant structure, and therefore extremely insusceptible to hydrolysis. Accessible cleavage reagents are often nonselective, some are toxic, require high fold molar excess and harsh conditions. Aforementioned reasons prompted the development of new, more efficient chemical proteases. Metal ion-assisted peptide bond hydrolysis is a favourable alternative for enzymatic cleavage of proteins with promising applications in biochemistry and bioengineering. Many metal ions and their complexes have been tested for such reactivity on a number of substrates, from dipeptides and oligopeptides to proteins. Among such, high efficiency was demonstrated by transition metal ions. In this study, we investigated the regioselectivity of peptide bond cleavage by various platinum(II) and palladium(II) complexes to determine how these metal ions, and steric factors direct proteolysis of bovine hemoglobin and human monoclonal antibody IgG1. Selective hydrolysis was accomplished by applying 10-fold molar excess of reagents, during 12-hour incubation, at pH 2.5 and 40 ᵒC. Product fragments were determined, based on the specific fragmentation pattern of their precursor ion, by tandem mass spectrometry. Thereat, platinum(II) complex with bidentate-coordinated ethylenediamine showed greater selectivity than the palladium(II) analog. For [Pt(en)(H2O)2]2+ 17 fragments were identified after cleaving hemoglobin sequence, apropos to 10 fragments for IgG1. In respect to [Pd(en)(H2O)2]2+, we assigned 34 product fragments to hemoglobin polypeptide chain, and 11 fragments to the sequence of IgG1. Regioselectivity of cleavage, determined by amino acids in the immediate vicinity, included side chains of l-methionine, l-cysteine and l-histidine. To a lesser extent, hydrolysis of bovine hemoglobin occurred near l-aspartic acid (hemoglobin) and non-polar l-glycine and l-tryptophan (IgG1). When we examined efficiency of more complex, dinuclear compounds bridged with aromatic pyrazine or pyridazine, here shown as [{M(en)(H2O)}2(-L)]4+, the efficacy and yield of hydrolysis were almost equal. Our results indicate the possibility of using platinum(II) and palladium(II) compounds for proteolysis, show reaction conditions in which hydrolysis occurs, and demonstrate selectivity of proteolysis as the basis for the application of these compounds in biochemical practice.