In this study, we present a first-of-a-kind biological-hardware-software tool to evaluate the physiological condition of in vitro myotubes in response to electrical stimulation. We demonstrate that impedance spectroscopy on a microelectrode array can testify for physiological changes of muscle cells under electrical stimulation. The platform is designed for simultaneous bioimpedance spectroscopy, electrical stimulation, and optical microscopy. It includes a microelectrode array, a custom hardware-software interface, and a commercial impedance analyzer. We used a well-established muscle cell model (C2C12) and developed a culture protocol suited for long-term recordings on microelectrode arrays. Electrical stimulation was applied with carbon electrodes and ad hoc electronics for current stimulation. Muscle cell bioimpedance measurement was complemented with optical microscopy video to record contractions. Then, the influence of electrical stimulation on the contractile activity of myotubes and on their bioimpedance was analyzed. Results validated the functionality of the hardware/software platform when used with our contractile muscle model. A bioimpedance-based metric was defined to evaluate changes in myotubes’ physiology. After playing multiple stimulation scenarios, analysis showed that the bioimpedance metric decreases as duration or frequency of stimulation increase.