Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, product sieving losses due to membrane fouling still limits the performance of these systems. We have obtained new insights into the underlying fouling phenomena using a combination of scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) to explore the nature and location of foulants, with the specific identity of individual proteins examined by matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). ATF experiments were performed using 0.2 µm polyethersulfone hollow fiber membranes with Chinese Hamster Ovary (CHO) cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet / exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. Individual proteins eluted from the fouled membranes showed multiple species commonly associated with extracellular vesicles, suggesting that deposition of these larger vesicles may be a critical contributor to membrane fouling. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.