In this paper we describe a new approach to measure pH differences in microfluidic devices and demonstrated acidification rate measurements in on-chip cell culture systems with nl wells. We use two miniaturized identical iridium oxide (IrOx) thin film electrodes (20 micromx400 microm), one as a quasi-reference electrode, the other as a sensing electrode, placed in two confluent compartments on chip. The IrOx electrodes were deposited onto microfabricated platinum (Pt) electrodes simultaneously using electrodeposition. Incorporating the electrodes into a microfluidic device allowed us to expose each electrode to a different solution with a pH difference of one pH unit maintaining a confluent connection between the electrodes. In this configuration, we obtained a reproducible voltage difference between the two IrOx thin film electrodes, which corresponds to the electrode sensitivities of -70 mV/pH at 22 degrees C. In order to measure the acidification rate of cells in nl cell culture volumes we placed one IrOx thin film electrode in the perfusion channel as a quasi-reference electrode and the other in the cell culture volume. We obtained an acidification rate of 0.19+/-0.02 pH/min for fibroblast cells using a stop flow protocol. These results show that we can use two identical miniaturized microfabricated IrOx electrodes to measure pH differences to monitor the metabolic activity of cell cultures on chip. Furthermore, our approach can also be applied in biosensor or bioanalytical applications.