Human milk is known to contain several proteases, but little is known about whether these enzymes are active, which proteins they cleave, and their relative contribution to milk protein digestion in vivo. This study analyzed the mass spectrometry-identified protein fragments found in pooled human milk by comparing their cleavage sites with the enzyme specificity patterns of an array of enzymes. The results indicate that several enzymes are actively taking part in the digestion of human milk proteins within the mammary gland, including plasmin and/or trypsin, elastase, cathepsin D, pepsin, chymotrypsin, a glutamyl endopeptidase-like enzyme, and proline endopeptidase. Two proteins were most affected by enzyme hydrolysis: β-casein and polymeric immunoglobulin receptor. In contrast, other highly abundant milk proteins such as α-lactalbumin and lactoferrin appear to have undergone no proteolytic cleavage. A peptide sequence containing a known antimicrobial peptide is released in breast milk by elastase and cathepsin D.