In this study we establish an electrochemical platform based on two dimensional (2D) pyrolytic carbon electrodes for in vitro analysis of osteoblast differentiation. Electrochemical impedance spectroscopy (EIS) was used to monitor cell adhesion and proliferation, while an electrochemical assay based on square wave voltammetry (SWV) was applied to measure the activity of the differentiation marker alkaline phosphatase (ALP). 2D pyrolytic carbon electrodes were fabricated and used to monitor Saos‐2 cell differentiation for a period of up to 21 days. With this method it was possible to detect a faster increase of ALP activity for cells cultured in medium supplemented with differentiation factors compared to cells cultured in growth medium. This was confirmed by the results obtained with Alizarin Red staining, showing that cells subjected to osteogenic medium went through the entire differentiation process, from proliferation to mineralization. Finally, for the first time, real‐time monitoring of ALP activity combined with continuous EIS monitoring of the same cell culture was achieved using the pyrolytic carbon electrodes.