Human milk is the truest form of personalized nutrition, supporting dynamic needs of the infant with nutritional and bioactive constituents that change throughout lactation. Additionally, human milk is individual specific and is unique for each mother-infant dyad. Proteins and endogenous peptides are 2 key classes of major human milk components making up the proteome, each with unique and synergistic functionality, providing protection for the healthy development of infants. Our objective was to comprehensively characterize and quantify the human milk proteome utilizing a personalized multiomics approach. We assessed in-depth individual variations of the human milk proteome across lactation, by mass spectrometry-based proteomics and peptidomics. The human milk proteome showed gradual changes primarily derived from the individual mother, and temporal changes over lactation. Additionally, a strong and rapid compositional change of the proteome was observed, albeit unexpectedly, resulting in an altered milk composition with indicated immune and inflammatory response functionality. Our personalized multiomics profiling approach resulted in the overall systematic annotation of the milk proteome reflecting the evolving development of the mammary gland and the maturing infant.