The characterisation of proteome and peptidome of adolescent mothers’ breast milk brings important information to both mother’s and infant’s health; however, it has not been investigated. Bioactive peptides derived from milk proteins have numerous functions. The bioactivity of breast milk peptides includes anti-inflammatory and antimicrobial activities and regulation of gastrointestinal function. We aimed to characterise the proteome and peptidome of mature breast milk of adolescent mothers and investigate whether it is affected by lactational period. We used a combination of electrophoretic and nano-scale LC-quadrupole time-of-flight MS/MS (nLC-Q-TOF-MS/MS) techniques and bioinformatics to explore the proteome of human skimmed milk expressed by lactating adolescents in two groups according to postpartum period (up to 3 and over 5 weeks postpartum). This is the first study that analysed the proteome of adolescent mothers’ breast milk produced during two periods of lactation using 1D-electrophoresis combined with nLC-Q-TOF-MS/MS analysis. Our results showed that the protein composition of adolescent milk varies independently of lactation stage and showed high inter-individual variation. A total of 424 proteins were identified in skimmed milk, of which 137 proteins were common to both groups. Most of the peptides found in adolescents’ breast milk were not derived from major proteins in milk. Association maps showed several interactions between groups of peptides that pointed to the relevance of breast milk peptides to neonatal defensive system.