Maximizing cell growth rate and cell yield are among the most important features of a successful mammalian cell culture production process. To minimize time and resources needed to scale up cell mass it is important to maintain the cultures in exponential growth at every scale. Here we report results comparing viable cell counts, packed cell volume, intracellular nucleotide ratios, cell cycle analysis, and on-line oxygen uptake rates (OUR) and optical density for the determination of the end of exponential growth to optimize transfer times during scale-up of CHO cell cultures. Viable cell concentration, packed cell volume, and relative abundance of cells in S-phase were not very reliable at determining the end of exponential growth during the process. In contrast, on-line determination of OUR and off-line determination of intracellular nucleotide ratios (U-ratio) were very sensitive to changes in growth rate, enabling clear determination of the end of exponential growth within a short time. Although on-line OUR was found to be the most convenient and fastest method, it is restricted to instrumented and continuously monitored cultures. In contrast the nucleotide method can be applied with any culture scale and condition but needs the availability of an operator running an HPLC system and takes about an hour from sampling to result. Optical density showed an inflection along with OUR and U-ratio but was less sensitive in determining the end of exponential growth.