Donor human milk (DHM) is recommended for preterm infants when a parent's own milk is unavailable or insufficient. To ensure microbiological safety, DHM is processed by donor milk processing companies using vat pasteurization (Vat-PT), ultra-high temperature (UHT) sterilization or retort (RTR) sterilization with and without homogenization. These processes may differentially affect the bioactive proteins in DHM. LC-MS/MS-based proteomics was applied to compare the detection of proteins in the soluble fraction of DHM after Vat-PT, UHT sterilization, RTR sterilization and homogenization in comparison with raw DHM (referred to herein as intact protein survival or retention). Protein retention (based on protein counts and abundances) was higher after Vat-PT than after UHT, and retention was higher after UHT sterilization than after RTR sterilization. Homogenization further decreased protein retention (based on protein counts and abundances) in both UHT- and RTR-treated samples. Representative DHM proteins including lactoferrin, lysozyme, immunoglobulins and caseins had higher retention after Vat-PT (5–57% decrease) than UHT (25–82% decrease) and RTR (54–88% decrease) treatments. These results will guide DHM processing to preserve bioactive proteins and potentially improve infant outcomes.