This paper describes a novel microfluidic-based assay for spinal cord injury (SCI) research. Conventional methods such as neurite outgrowth and strip assays cannot recapitulate the organized structure of the spinal cord and thus poorly simulate the injury microenvironment. In addition, it is difficult to obtain quantitative results to compare subtle differences on a chemical's effect on normal growth and regeneration. In SCI, the cell bodies are often located away from the immediate lesion, while the damaged and regenerating axons are exposed to the inhibitory milieu of the scar-tissue. We combined micropatterning and microfluidics to selectively place high purity CNS neurons on favorable substrate but allow only axons to interact with permissive (i.e. polylysine) and inhibitory substrates (i.e. aggrecan) presented in alternating strips. On patterned surfaces, axons were confined on permissive lanes and consistently avoided inhibitory strips. Since processes are expected to proceed in a pre-defined direction/geometry, even small deviations, indicative of the drug's effectiveness, can be readily detected. To demonstrate the potential utility of the method in drug screening for SCI, we used chondroitinase-ABC as a model drug to overcome the inhibitory effects of aggrecan. Enzymatic treatment promoted axons to cross onto the nerve-inhibitory strips and extend randomly across the pattern. Such effects can be easily observed and confidently quantitated to obtain objective comparison. This approach is amenable for high throughput screening and may be used to study the effects of pharmaceuticals that suppress inhibitors of neuronal growth/regeneration.