Transepithelial electrical resistance (TEER) measurement is a label free, rapid and real-time technique, which is commonly used to evaluate the integrity of cell barriers. TEER characterization is important for applications, such as tissue (brain, intestines, lungs) barrier modeling, drug screening, and cell growth monitoring. Traditional TEER methods usually only show the average impedance of the whole cell layer, and lack accuracy and the characterization of internal spatial differences within cell layer regions. Here, we introduce a new spatial TEER strategy that utilizes microelectrode arrays (MEA) integrated in a Transwell to dynamically monitor TEER. A new electrical model which could reveal spatial impedance non-uniformity was proposed to extract accurate resistance from the measured data. Based on our method, the TEER signals from 16 different regions were successfully monitored in real time. The mapped impedance hotspots in different regions closely correlate with both fluorescence cell staining signals and calculated cell coverage, indicating the effectiveness of the developed spatial TEER system in monitoring local cell growth in vitro. The real-time spatial TEER responses to ethylene glycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and cisplatin were studied, which could either reduce barrier integrity or inhibit cellular growth. The obtained results demonstrated the spatial TEER's applicability for cell barrier function and cell growth monitoring. Our approach provides accurate spatial electrical information of cell barriers and holds potential applications in drug development and screening.