Human milk not only contains all nutritional elements that an infant requires, but is also the source of components whose regulatory role was confirmed by demonstrating health-related deficiencies in formula-fed children. A human milk diet is especially important for premature babies in the neonatal intensive care unit (NICU). In cases where breastfeeding is not possible and the mother’s own milk is insufficient in volume, the most preferred food is pasteurized donor milk. The number of human milk banks has increased recently but their technical infrastructure is continuously developing. Heat treatment at a low temperature and long time, also known as holder pasteurization (62.5◦C, 30 min), is the most widespread method of human milk processing, whose effects on the quality of donor milk is well documented. Holder pasteurization destroys vegetative forms of bacteria and most viruses including human immunodeficiency virus (HIV) herpes and cytomegalovirus (CMV). The macronutrients remain relatively intact but various beneficial components are destroyed completely or compromised. Enzymes and immune cells are the most heat sensitive elements. The bactericidal capacity of heat-pasteurized milk is lower than that of untreated milk. The aim of the study was for a comprehensive comparison of currently tested methods of improving the preservation stage. Innovative techniques of milk processing should minimize the risk of milk-borne infections and preserve the bioactivity of this complex biological fluid better than the holder method. In the present paper, the most promising thermal pasteurization condition (72◦C–75◦C,) and a few non-thermal processes were discussed (high pressure processing, microwave irradiation). This narrative review presents an overview of methods of human milk preservation that have been explored to improve the safety and quality of donor milk.