In vitro analysis requires cell proliferation in conditions close to physiological. Lab-on-a-Chip (LoC) devices simplify, miniaturize and automate traditional protocols, with the advantage of being cheaper and faster thanks to the shorter diffusion distances. Main limitation of current LoCs is still to control the culture conditions. Most of LoC employ off-chip equipment to determine cell culture activity, which confers a limited monitoring capacity. The few systems integrating transducers on-chip present important functional problems mostly associated to the attachment of biomolecules to the transducer surface (i.e. biofouling) and the impossibility to re-calibrate the sensors during cell culturing. This limitation is settled in the present LoC containing a network of micro-channels and micro-chambers, which allows: (i) cell seeding and cultivation avoiding biofouling risk, (ii) multiplexed analysis of cell culture, reactivation and recalibration of the (bio)sensors without compromising cells viability, (iii) cells imaging and (iv) reference electrode compartmentalization to guarantee its stability. The activity of the culture is monitored with four independent electrochemical micro-electrodes for glucose, hydrogen peroxide, conductivity and oxidation reduction potential. Electrochemical analysis is complemented with high-resolution confocal microscopy analysis. This paper demonstrates the suitability of the current configuration for cell culture monitoring and future application to drug screening or organ-on-a-chip development. This article is Open Access Please wait while we load your content... Something went wrong. Try again?