Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease with minimal treatment options and short survival time for patients beyond symptom onset. Early disease pathogenesis includes denervation at the neuromuscular junction (NMJ), dying-back axonopathy, and distal axon degeneration. RNASeq studies have been done looking for differences in gene expression profiles between iPSC-derived motor neurons from patients with ALS disease-causing mutations and controls, as these may give key insights into disease mechanisms. However, given early disease pathology, axonal gene expression specifically may contribute to disease. As such, we’ve focused this work on isolating axonal RNA. In most commonly used culture methods, neurons are grown on a dish or plate with axons extending out from neuronal body clusters in overlapping patterns, making it difficult to track long axons and study the distal axon. We have successfully cultured induced pluripotent stem cell (iPSC)-derived motor neurons in microfluidic devices that allow us to isolate the distal axons from the cell bodies. By using microfluidic devices with a double set of microchannels rather than single, we have been able to isolate pure populations of axons without contamination from cell bodies. We have also been able to extract high quality control RNA for sequencing from the spatially separated compartments, allowing us to analyze axonal RNA expression profiles independent of neuronal cell body expression. With this work we have set up an experimental system to isolate pure axons and study their gene expression in the context of ALS-disease causing mutations.