At high viable cell concentrations in large-scale mammalian cell culture processes, the accumulation of dissolved carbon dioxide (dCO2, typically quantified as an equilibrium gas-phase concentration) becomes problematic as a result of low CO2 removal rates at reduced surface-to-volume ratios. High dCO2 concentrations have previously been shown to inhibit cell growth and product formation in mammalian cells and to alter the glycosylation pattern of recombinant proteins. Therefore, reliable monitoring and control of dCO2 are important for successful large-scale operation. Off-line measurements by instruments such as blood gas analyzers (BGA) are constrained by the low frequency of data collection and cannot be used for on-line control. In a preliminary evaluation of the YSI 8500 in situ sensor, a response time (t90%) of 6 min, sensitivity of 0.5% CO2 (3.6 mmHg), and linearity of measurement (R2 = 0.9997) between the equivalent gas-phase partial pressure of 0−180 mmHg (0% and 25% CO2) were established. Measurements were found to be unaffected by culture pH and typical mammalian cell culture concentrations of glucose, glutamine, glutamate, lactate, and ammonium. The sensor withstood repeated sterilization and cleaning cycles. The reliability of this sensor was demonstrated in microcarrier-based Chinese hamster ovary (CHO) cell perfusion cultures at reactor scales of 30, 40, 340, and 2000 L and was successfully implemented in a dCO2 control strategy using N2 sparging.