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Neuronal processes have an RNA composition that is distinct from the cell body. Therefore, to fully understand neuronal biology in health and disease we need to study both somas, dendrites and axons. Here we describe a detailed protocol of a newly refined method, Axon-seq, for RNA sequencing of axons (and dendrites) grown in isolation using single microfluidic devices. We also detail how to generate motor neurons from mouse and human pluripotent stem cells for sequencing, but Axon-seq is applicable to any neuronal cell. In Axon-seq, the axons are recruited through a growth factor gradient, lysed and directly processed to cDNA without RNA isolation. A careful bioinformatic step ensures that any soma-contaminated samples are easily identified and removed.