The bioprocess development cycle is a complex task that requires a complete understanding of the engineering of the process (e.g., mass transfer, mixing, CO2 removal, process monitoring, and control) and its affect on cell biology and product quality. Despite their widespread use in bioprocess development, spinner flasks generally lack engineering characterization of critical physical parameters such as kLa, P/V, or mixing time. In this study, mass transfer characterization of a 250-mL spinner flask using optical patch-based sensors is presented. The results quantitatively show the effect of the impeller type, liquid filling volume, and agitation speed on the volumetric mass transfer coefficient (kLa) in a 250-mL spinner flask, and how they can be manipulated to match mass transfer capability at large culture devices. Thus, process understanding in spinner flasks can be improved, and these devices can be seamlessly integrated in a rational scale-up strategy from cell thawing to bench-scale bioreactors (and beyond) in biomanufacturing. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.