Many pathogens, including viruses, bacteria, as well as bacterial toxins, enter their target cells by endocytosis leading to accumulation of pathogenic and cellular proteins in endosomes. Here, we present detailed experimental instructions on isolation of endosomes after virus infection and their subsequent biomolecular characterization. The isolation of endosomes is based on discontinuous sucrose gradient centrifugation, where different endosomal compartments accumulate at a specific sucrose interface. This enables the enrichment and separation of the virus-interacting and co-internalized cell-surface receptors and membrane-associated proteins. The endosomal fractions can be further analyzed by Western blot or quantitative real-time PCR, which reveals changes in the viral protein or DNA content during the processes of endocytosis and endosomal escape. In addition, comparative quantitative mass spectrometry enables the identification of unknown host-cell factors required for infection.