Consumption of bioactive milk proteins enhances preterm infant development. Preterm infants are often supplemented with donor human milk (DHM) when parents’ own milk is unavailable in sufficient quantity. DHM is normally Holder pasteurized (HoP) to inactivate pathogens; however, it denatures many bioactive proteins. Alternatively, ultraviolet-C (UV–C) irradiation can inactivate pathogens while preserving milk proteins. This study examined the effects of HoP and UV–C on the proteome of DHM (n = 1 pool, processing in triplicate). Protein counts in HoP samples (n = 198) were lower than in raw DHM (n = 228) and UV–C at 6000 J/L (n = 239) samples. HoP resulted in significant reductions in relative abundance for 80 proteins (p-values <0.05, >0.5-fold decrease), whereas UV–C at 6000 J/L resulted in only 5 proteins significantly decreasing. Proteins reduced by HoP that were preserved by UV–C treatment had a broad array of functions, including antimicrobial, iron-binding, immunomodulatory, and proteolytic. Applying UV–C processing for donor milk processing could improve outcomes in preterm infants.