Bovine hemoglobin was hydrolyzed with pepsin in a batch stirred tank reactor; the resulting peptides were identified, in a time dependent and comprehensive manner, using reversed phase-high performance liquid chromatography (RP-HPLC) coupled with electrospray ionization tandem mass spectrometry (ESI-MS/MS) by means of database searching. Peptic digestion of bovine hemoglobin yields more hydrophobic peptides at a low degree of hydrolysis, and more hydrophilic peptides at a later stage of hydrolysis. The release kinetics of the bioactive peptides was also followed based on the RP-HPLC profiles. In addition, the behavior of peptic digestion of hemoglobin alpha and beta chains was compared in terms of profiling the identified peptides. Thirty-two peptides were recovered by a process of hydrolysis from the alpha chain of the peptide; whereas the corresponding result for the beta chain was 19 peptides with around 67% sequence coverage. The main factor responsible for non-peptic susceptibility of the central region of the beta chain was their relatively higher hydrophilicity.