Electrical Impedance Spectroscopy (EIS) is a non-invasive technique that allows monitoring of cancer cells and cancer tissue. EIS may be performed by two electrode-technique (2-ET) where both electrodes are used for excitation and recording purposes, or by four electrode-technique (4-ET) where the two outer electrodes are used for current excitation and the two inner electrodes record the resulting signal.1 The amplitudes of the impedances at specific frequencies (1-10 KHz) are typically used to identify differences between adherent cells, tissues and bare electrodes. Here we successfully used both techniques to closely monitor the adhesion of tumor spheroids using EIS. Three-Dimensional Tumor Spheroids (3D-TS, diameter 200–500 µm) made of human colon cancer cell line HCT 116 were cultured on a Micro Electrode Array (MEA; electrode spacing: 70 µm). EIS was performed at three time points (4h, 21h, 66h) after interfacing the 3D-TS to the MEA. Using the MEA electrode map, we visually selected pairs of electrodes that are covered with 3D-TS. The impedance magnitude and phase shift were analyzed using custom-written scripts (Python). Both techniques (2-ET and 4-ET) allow for reliable, noninvasive identification of adherent spheroids. The identification was confirmed using optical microscopy. In 2-ET measurements, we obtained significantly higher impedance amplitude for electrodes underneath the tumor spheroids as compared to bare electrodes. In 4-ET measurements, the impedance amplitudes are reduced but well distinguishable from control measurements. The 2-ET facilitates the generation of a 2D heat map, which closely mimics the size of the TS and its position on the MEA. In 2-ET, the impedances of the electrodes influence the measurement, and the measured signal corresponds to the series connection of the spheroid and electrodes. However, In 4-ET, the sensing electrodes don’t carry any current thanks to the high input impedance of the amplifier and prevent their effect on the measurement. Ongoing experiments are performed to test the effect of anticancer drugs, and results will be presented. 1. Bragos, R. et al.”, Four Versus Two-Electrode Measurement Strategies for Cell Growing and Differentiation Monitoring Using Electrical Impedance Spectroscopy,” IEEE EMBS. pp:2106-2109, 2006.