Microfiltration membranes are increasingly used to retain the cells inside bioreactors while continuous harvest of the desired biopharmaceutical occurs in perfusion processes. One method of microfiltration is Alternating Tangential Flow (ATF) filtration, which involves moving the cultivation broth tangentially back and forth across the membrane, which results in a backwash effect that reduces fouling. In this study, fouling was investigated with asymmetric polysulfone hollow fibers operated in ATF mode attached to a bioreactor producing a recombinant protein in Chinese Hamster Ovary (CHO) cells. Fouling was assessed through different approaches, including determination of critical flux using an improved flux-step method. Fouling was studied through measurements of Transmembrane Pressure (TMP), protein transmission, membrane pore size and staining of the membrane after operation to visualize the distribution of biological fouling inside the membrane. For critical flux determination, fluxes of up to 69 LMH were used without exceeding the critical flux. No sign of fouling was observed for the short-term (<3.5 h) critical flux experiment. However, during prolonged operation at 8.3 LMH the TMP jumped to 0.9–0.95 bar, indicating fouling. At this state, the protein transmission remains at the same high level (>88 %). Display omitted •Mammalian cell culturing for production of recombinant protein.•Reverse asymmetric membrane hollow fiber filtration in perfusion process.•Critical flux determination in Alternating Tangential Flow Filtration.•Distribution of biological fouling in membrane by staining techniques.•Constantly high protein transmission even though significant fouling is present.