Hundreds of naturally occurring milk peptides have been found in term human milk. Whether there are differences between the levels of these peptides between term and preterm milk remains unknown. Premature milk is produced before complete maturation of the mammary gland and is under the influence of a different hormonal milieu, which could change enzymatic activity and protein expression within the mammary gland and result in an altered peptide profile. We employed nano-liquid chromatography tandem mass spectrometry to identify naturally occurring peptides in term and premature milks at multiple time-points across lactation and compare the abundances of these peptides. We found that preterm milks produced more unique peptide sequences than term milks on average (359 vs. 286). The peptides identified were searched for overlapping sequences with an in-house database of known functional peptides. Specifically, we found that both term and preterm milks contain peptides overlapping with known sequences with antimicrobial, opioid antagonist and immunomodulatory actions. We also compared the enzymes involved in degradation of both milks via bioinformatic analysis. This analysis reveals that plasmin is more active in preterm milk than term milk.