In this protocol, we describe steps to design, fabricate and use the Device for Axon and Cancer cell Interaction Testing (DACIT) in 2D and in 3D. In the first section, we detail steps to generate the mask, the master and the smooth-on mold. Next, we describe the step-by-step protocol for fabricating the DACIT, loading sensory neurons and cancer cells in 2D or 3D. We compare axonogenesis using PC-12 cell line and primary embryonic or adult sensory neurons, demonstrating the superior neurite growth in primary cells. We demonstrate DACIT can be used to compartmentalize neuronal soma and axons and expose them to different conditions, or to form a temporary gradient of neurotransmitter. Finally, we show that DACIT can be used to measure spheroid invasion in 3D in the presence of axons.