Chemotaxis is the phenomenon by which the migration and invasion of cells is directed in response to an extracellular chemical gradient. Chemotaxis of tumor cells and tumor-associated inflammatory and stromal cells is mediated by chemokines, chemokine receptors, growth factors, and growth factor receptors. Current techniques used to study chemotactic driven cell invasion and metastasis utilize two-dimensional migration assays involving imaging and analyzing tumor cells on glass slides or plastic surfaces, which requires large numbers of cells and often lacks real-time monitoring of vertical cell movement and systematically controlled chemotactic gradients, leading to contradictory results compared to those from clinical investigations and animal models. We addressed such challenges by developing a high-throughput microdevice with 4000 ultraminiaturized wells to monitor real-time, three-dimensional cell invasion over a wide range of cell densities and also screen drugs that inhibit cell invasion and potentially prevent metastatic malignancy. Additionally, this microdevice generates opposing gradients for two types of cells on the same chip, which builds a controlled system with sequentially changing components to study environmental effects from basal and immune cells.