In the biopharmaceutical industry, accurate prediction of the oxygen uptake rate (OUR) by the cells is critical to understanding cell health. Accurate estimation of the oxygen transfer rate (OTR) presents a significant challenge in predicting OUR, and OTR is dependent on the volumetric oxygen mass transfer coefficient (kLa). Often kLa is assumed to be constant throughout a fermentation and estimated from experiments using no cells and in a buffer solutions. Yet, it is well-known that additions (i.e., glucose, base, and antifoam), cell secretions, and stir speed markedly effect kLa. Currently, there are three standard methods used to estimate kLa; however, all three have significant issues. This rapid communication describes a novel, non-disruptive method to estimate kLa by modulating the gas flow rate. This approach allowed accurate kLa estimates at multiple times throughout Escherichia coli batch fermentations using a non-disruptive technique, thus lowering the risk of losing a fermentation due to inadequate oxygenation.