Distal axon degeneration, dying-back, is a hallmark of motor neuron diseases, such as ALS, that precedes symptom onset and motor neuron death both in human patients and animal models. While motor neurons derived from human iPSCs (hMNs) hold promise for advancing ALS research, the length of axons, regenerative capacity, and mutant-specific innervation of neuromuscular junctions (NMJs) by these human neurons is not well-characterized. hMNs cluster into circular groups as they grow, and extend axons to other clusters, confounding quantification of axon outgrowth from individual hMNs. To address this, we have cultured hMNs from ALS patients and controls in custom microfluidic devices, and sequestered neuronal cell bodies in the main compartment that extended processes through microgrooves into two adjacent axonal compartments. We determined that devices with ample room in the axonal compartments are appropriate for examining axonal outgrowth, and allow for individual tracing of axons that are millimeters in length. We are able to sever axons at the entry point to the axonal compartments, and use time-lapse live imaging to quantify regeneration speed. This system lays the groundwork for introducing relevant cell types and gathering electrophysiological data from myocytes innervated by hMNs. We are now exploring the introduction of relevant human cell types, such as skeletal myocytes, into the axonal compartment in order to study ALS mutation-specific effects on structural and functional innervation of NMJs.