Abstract Electrical impedance spectroscopy (EIS) measurements with four electrodes is widely used in the study of electrical properties of cells in suspension. Under less than ideal conditions, this set up enables the removal the surface polarization effects. However, in the case of HeLa cells in suspension, we noted that the electrical impedance spectra reproducibility is affected by the impurities and ionic species adsorbed on the electrode surface. We studied the influence of the electrode’s surface conditions on the EIS measurements of HeLa cells in suspension. EIS spectra were obtained before and after submitting the electrodes to a cleaning process. Chronopotentiometry and open circuit potential (OCP) measurements were carried out to verify the electrode’s surface conditions. The main effect of cleaning the electrodes was to decrease the standard deviation of the EIS data (On average, 3.42? to 0.70? for PBS and 17.22? to 0.31? for PBS with HeLa cells). OCP measurements evidenced surface differences between the cleaned and uncleaned electrodes. In addition, the chronopotentiometric curve obtained for the gold electrode/PBS system showed the adsorption of chloride and phosphate ions and other species existing in PBS on the electrode surface at 0.28 V and 0.53 V. Results suggest that the adsorbed species on the electrode surface led to an electrostatic build-up, which has a non-passive behavior.