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. We have performed axotomies and compared regeneration speed of hMNs harboring ALS-linked mutations, including hMNs with a SOD1A4V mutation to an isogenic corrected control. In co-cultures with primary human myoblast-derived myofibers, hMNs form NMJs. This system lays the groundwork for gathering electrophysiological data from myocytes innervated by hMNs in the axonal compartment, and introducing relevant cell types. Systematic permutations of this microfluidic culture system have the potential to elucidate the ALS mutation-specific effects on axonal regeneration and structural and functional innervation of NMJs.