Current preclinical models of neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS), can significantly benefit from in vitro neuroengineering approaches that enable the selective study and manipulation of neurons, networks, and functional units of interest. Custom-designed compartmentalized microfluidic culture systems enable the co-culture of different relevant cell types in interconnected but fluidically isolated microenvironments. Such systems can thus be applied for ALS disease modeling, as they enable the recapitulation and study of neuromuscular junctions (NMJ) through co-culturing of motor neurons and muscle cells in separate, but interconnected compartments. These in vitro systems are particularly relevant for investigations of mechanistic aspects of the ALS pathological cascade in engineered NMJ, as progressive loss of NMJ functionality may constitute one of the hallmarks of disease related pathology at early onset, in line with the dying back hypothesis. In such models, ability to test whether motor neuron degeneration in ALS starts at the nerve terminal or at the NMJ and retrogradely progresses to the motor neuron cell body largely relies on robust methods for verification of engineered NMJ functionality. In this study, we demonstrate the functionality of engineered NMJs within a microfluidic chip with a differentially perturbable microenvironment using a designer pseudotyped ΔG-rabies virus for retrograde monosynaptic tracing.