AbstractCell invasion/migration through three‐dimensional (3D) tissues is not only essential for physiological/pathological processes, but a hallmark of cancer malignancy. However, how to quantify spatiotemporal dynamics of 3D cell migration/invasion is challenging. Here, this work reports a 3D cell invasion/migration assay (3D‐CIMA) based on electromechanical coupling chip systems, which can monitor spatiotemporal dynamics of 3D cell invasion/migration in a real‐time, label‐free, nondestructive, and high‐throughput way. In combination with 3D topological networks and complex impedance detection technology, this work shows that 3D‐CIMA can quantitively characterize collective invasion/migration dynamics of cancer cells in 3D extracellular matrix (ECM) with controllable biophysical/biomechanical properties. More importantly, this work further reveals that it has the capability to not only carry out quantitative evaluation of anti‐tumor drugs in 3D microenvironments that minimize the impact of cell culture dimensions, but also grade clinical cancer specimens. The proposed 3D‐CIMA offers a new quantitative methodology for investigating cell interactions with 3D extracellular microenvironments, which has potential applications in various fields like mechanobiology, drug screening, and even precision medicine.