Good electronic and optical properties (transmittance: >83%) make indium tin oxide (ITO) an attractive electrode substrate. Despite the commercial availability of high-quality ITO and some low-cost methods for direct deposition being in use by now, the definition of patterns is still a concern. Putting their popularity and extensive use aside, the manufacturing of ITO electrodes so far lacks a rapid, highly reproducible, flexible, cost-effective, easy patterning process that could surpass difficult, time-consuming techniques such as lithography. Herein, we present a low-cost method based on CO₂ laser irradiation for preparing ITO microelectrodes and electrode arrays. Electrodes of different sizes and shapes were examined to identify the performance of the proposed methods. Direct ablation of the ITO layer was optimized for rectangular electrodes of 25, 50, and 100 μm in width, while laser cutting of scotch tape stencils and subsequent wet etching were used to create circular electrodes with a diameter of 1.75 mm. A multielectrode array system consisting 8 of these circular electrodes was fabricated on a (25 × 25) mm² plate, characterized electrochemically through cyclic voltammetry (CV) and scanning electrochemical microscopy (SECM), and as an example application used for monitoring the anchoring behavior of HeLa and HepG2 cell cultures through cell-based electrochemical impedance spectroscopy. Together, the direct ablation method and preparation of laser cut stencils form a complete toolbox, which allows for low-cost and fast fabrication of ITO electrodes for a wide variety of applications. To demonstrate the general availability of the method, we have also prepared a batch of electrodes using a laser plotter in a local printing shop, achieving high intra-workshop reproducibility.