Impedance measurement using Electrochemical Impedance Spectroscopy is a widely utilized technique in neural electrodes. Research and clinical devices that incorporate stimulating and recording microelectrodes routinely characterize the material's integrity and its functionality through impedance measurement. Nominal impedance values ensure a stable neural electrode-tissue contact capable of passing through power efficient electric signals with desired signal-tonoise ratio or effective volume coverage. However, the complexity of the in vivo environment limits the usage of the three-electrode setup, which has been accepted as the ideal method in providing a stable impedance measurement. Impedance data measured from microelectrodes in threeelectrode and two-electrode setups show that the two setups have similar outcomes in terms of the impedance modulus over a 0.5 Hz-100 kHz frequency range. Usage of a platinum counter electrode lowered the overall variance in impedance readings compared to the stainless steel counter electrode. However, correlation coefficient values $(>0.97)$ between three-electrode and two-electrode setups show that impedance values seldom deviate due to changes in electrode setup. Based on the results of this study, the usage of the two-electrode setup in vivo allowed acceptable electrochemical impedance spectroscopy accuracy, and the utilization of a platinum counter electrode is recommended to reduce measurement variance.