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This article belongs to the Special Issue Novel Approaches for Developing and Characterizing Value-Added Applications of Food and Agricultural Byproducts., Recent studies have demonstrated the positive effects of regular intake of milk fat globule membranes (MFGMs) on neural and cognitive development, as well as immune and gastrointestinal health in infants and elders. Dairy products and by-products generated from the butter and butter oil manufacturing process are valuable sources of MFGM. Thus, in view of the growing need to reduce by-products and waste, it is crucial to foster research aimed at the valorization of dairy by-products rich in MFGM. For this purpose, all the by-products coming from butter and butter oil production (from raw milk to the related by-products) were used to study the MFGM isolated fractions, followed by their characterization through a combined lipidomic and proteomic approach. The patterns of polar lipids and proteins indicated that buttermilk (BM), butterserum (BS), and their mix (BM-BS blend) are the most suitable by-products to be employed as starting material for the isolation and purification of MFGMs, thus obtaining MFGM-enriched ingredients for the manufacture of products with high biological activity., This research was funded by the Spanish Ministry of Science, Innovation and Universities and Spanish Ministry of Science and Innovation. Projects: PDI2020-114821RB-I00 and PDC2021-121528-I00 MCIN/AEI//FEDER, UE.