Isolation and characterization of antigen-containing endosomes remains difficult utilizing standard purification techniques. Here, we describe a method, which allows isolation of antigen-loaded endosomes, that is based on flow cytometrical analysis and sorting. We specifically isolated antigen-containing endosomes from cells that had taken up fluorochrome-labeled ovalbumin via mannose receptor-mediated endocytosis. The protocol described here allows for the isolation of pure fractions of ovalbumin-containing endosomes and the extraction of proteins from these endosomes for analysis by western blot. Importantly, this protocol can be easily extended to other fluorochrome-labeled antigens and to primary antigen-presenting cells like bone marrow-derived dendritic cells.