Neuromuscular junctions (NMJs) are specialized synapses that mediate communication between motor neurons and skeletal muscles and are essential for movement. The degeneration of this system can lead to symptoms observed in neuromuscular and motor neuron diseases. Studying these synapses and their degeneration has proven challenging. Prior NMJ studies heavily relied upon the use of mouse, chick, or isolated primary human cells, which have demonstrated limited fidelity for disease modeling. To enable the study of NMJ dysfunction and model genetic diseases, we, and others, have developed methods to generate human NMJs from pluripotent stem cells (PSCs), embryonic stem cells, and induced pluripotent stem cells. However, published studies have highlighted technical limitations associated with these complex in vitro NMJ models. In this study, we developed a robust PSC-derived motor neuron and skeletal muscle co-culture method, and demonstrated its sensitivity in modeling motor neuron disease. Our method spontaneously and reproducibly forms human NMJs. We developed multiwell-multielectrode array (MEA) parameters to quantify the activity of PSC-derived skeletal muscles, as well as measured the electrophysiological activity of functional human PSC-derived NMJs. We further leveraged our method to morphologically and functionally assess NMJs from the familial amyotrophic lateral sclerosis (fALS) PSCs, C9orf72 hexanucleotide (G4C2)n repeat expansion (HRE), SOD1 A5V , and TDP43 G298S to define the reproducibility and sensitivity of our system. We observed a significant decrease in the numbers and activity of PSC-derived NMJs developed from the different ALS lines compared to their respective controls. Furthermore, we evaluated a therapeutic candidate undergoing clinical trials and observed a variant-dependent rescue of functionality of NMJs. Our newly developed method provides a platform for the systematic investigation of genetic causes of NMJ neurodegeneration and highlights the need for therapeutic avenues to consider patient genotype.