The separation of peptides from human blood and the degradation of hemoglobin was investigated by high-performance liquid chromatography (HPLC). From the experimental results, we determined the optimum mobile phase condition and gradient mode for the separation of peptides from human blood and identified five peptides. Four of the identified peptides were biologically active. The results show that some peptides form and accumulate during the storage of human blood. The kinetics of peptide formation was modeled to that of a first-order reaction. The probability of the short peptides being formed inside human erythrocytes was evaluated by analyzing the peptides using a non-cell model systems (in vitro).