Virus safety of fetal bovine serum (FBS) is a critical issue for cell culture and clinical applications of cell therapies. The size exclusion filtration of FBS-supplemented cell culture media through small-size virus retentive filter paper is presented to investigate its effect on cell culture. A substantial proportion of proteins (ca. 45%) was removed by nanofiltration, yet important transport proteins (albumin, fetuins, macroglobulins, transferrin) were unaffected. The cell viability of Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK-293) cells that were grown in media supplemented with nanofiltered FBS was surprisingly high, despite the observed protein losses. Protein depletion following nanofiltration resulted in detectable levels of autophagy markers.