Subcellular compartments of highly polarized cells, such as the cell bodies and axons of motor neurons, have unique transcriptomes to support local translation, providing axons with the autonomy to quickly react to stimuli. Culturing iPSC-derived motor neurons in Campenot Chambers enables isolation of RNA from axons and cell bodies in vitro followed by quantification of their unique transcriptomic profiles. The low yield of axonal RNA obtained per chamber poses a significant barrier to generating sequencing libraries. Here we describe an optimized version of RNA isolation and library preparation based on Shishkin et al. using readily available reagents. Using this optimized method, cell body and axon-derived samples exhibited a greater percentage of uniquely mapped reads, and a lower percent of mapping to repetitive regions and multiple loci compared to commercial kits. In addition to the technical advancement this method provides, it has the potential to facilitate translational research. Axonal RNA localization has relevance in the neurodegenerative field based on the observation that many ALS-associated mutations occur within RNA-binding proteins that are believed to function in transporting mRNA from the cell body to the axon [1-3]. Additionally, our preliminary analysis of ALS-associated mutant cell lines suggest that application of this method to in vitro ALS models will provide critical insight to our understanding of the pathology of motor neuron disease.