Production of biopharmaceuticals from mammalian cells requires generation of master, working and post-production cell banks of high quality under GMP conditions. An optimal cryopreservation strategy is needed for each new production cell line, particularly with regard to establishing production processes that are completely devoid of serum or even any animal components and to ensuring robust thaw performance for reliable production. Here, we describe a novel strategy employing flow-cytometric (FC) analysis of Annexin V-stained cells for high-throughput characterization of cell banks. Our data show that this method enables predictive evaluation of a cryopreservation strategy as early as 6h after thawing of cells. Furthermore, a broad study is presented characterizing various factors that may influence the quality of serum-free production cell banks from NSO and CHO cell lines. These results demonstrate how FC-based analysis can be used for development of future state-of-the-art cryopreservation strategies.