Human in vitro models of the neuromuscular junction (NMJ) are currently moving from embryonic stem cells to induced Pluripotent Stem Cells (iPSCs). With this, a robust model could be optimised for physiology and pathophysiology studies, as well as representing a drug screening platform. For this reason, the work presented here represents the optimisation of a human co-culture model of skeletal muscle (hSkM)/ iPSC-derived motor neurons (MNs) both in monolayer and in 3D tissue engineering collagen constructs. Firstly, human iPSC-derived motor neurons (MNs) were characterised over a period of 35 days to test their cholinergic potential. Then, primary human skeletal muscle (hSkM) and MNs were co-cultured on different substrates (gelatin and SureBond+ReadySet (Axol Bioscience)) and differentiated in various combinations of media to allow both myotube formation and neurite extension. Morphological (β-III Tubulin and Rhodamine Phalloidin) and interaction (α-Bungarotoxin and Synaptic Vesicle 2) immunofluorescent stainings were used to evaluate cell differentiation and co-localisation of pre and post-synaptic markers. Results from this study showed that the MNs presented a cholinergic phenotype up to 21 days; hSkM and MNs co-existed in culture and differentiated in neuronal Maintenance Medium (MM, Axol Bioscience); the 3D constructs allowed alignment and maturation of the muscle tissue, while providing a matrix for neurite extension and NMJ formation. This model has the potential to become a valid tool for in vitro drug screening while reducing the use of animals in research and providing the scientific community with a platform for personalised medicine.