In mammalian cell culture, especially in pharmaceutical manufacturing, pH is a critical process parameter that has to be controlled as accurately as possible. Not only does pH directly affect cell culture performance, ensuring a comparable pH is also crucial for scaling and transfer of processes. A sample-based offline pH measurement is commonly used to ensure correct bioreactor pH probe signals after sterilization and as a detection measure for drifts of probe signals. However, the sample-based pH offline measurement does not necessarily deliver required accuracy. Offsets between bioreactor pH and sample pH heavily depend on equipment, local procedures and the offline measurement method that is used. This article adequately describes a novel, non-invasive method to determine pH and pCO2 in sterile bioreactors without the need to sample and measure offline. This method utilizes the chemical correlation between carbon dioxide in the gas phase, dissolved carbon dioxide, bicarbonate and dependent proton concentrations that directly affect the pH in carbonate buffered systems. The proposed carbon dioxide-based pH reference method thereby is able to accurately determine the true pH in the bioreactor without the need to sample. The proposed method is independent of scale and bioreactor configuration and does not depend on local procedures that may differ between sites, scales or operators. Applicability of the method for both stainless steel and single use bioreactors is shown. Furthermore, the very same principles are applicable for non-invasive, online pCO2 monitoring.