The intracellular pH (pH(i)) is an important factor in the regulation of different cellular processes. It might therefore be used as a marker of the physiological state of cells cultivated in a bioreactor environment. We developed and validated therefore a methodology that permits a reproducible and reliable pHi measurement under such bioreactor culture conditions, contrary to earlier reported measurements, carried out on cells resuspended in buffers under nongrowth conditions. The hybridoma cells were sampled from the culture, stained with the pH-sensitive dye BCECF-AM BCECF = 2＇,7＇-bis-carboxyethyl-5,6-carboxyfluorescein and analyzed by flow cytometry. Such a measurement is perfectible to changes of the cells between the moment of sampling and of final analysis on the flow cytometer. All intermittent steps were for this reason studied in detail, either to determine the optimal conditions to be used or to characterize their influence on the final pH(i) value measured. Additional experiments were carried out, showing the representativeness of the measured pH(i) value for the pH(i) the cells possess really in the culture at the moment of sampling.