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Many traumatic, neurodegenerative, and neuroimmunological diseases are classically believed to mainly affect either the neuronal or the muscle side of the NMJ, and treatment options are lacking. Recent advances in novel techniques have helped develop in vitro physiological and pathophysiological models of the NMJ as well as enable precise control and evaluation of its functions. This paper reviews the recent developments in in vitro NMJ models with 2D or 3D cultures, from organ\u2010on\u2010a\u2010chip and organoids to biohybrid robotics. Related derivative techniques are introduced for functional analysis of the NMJ, such as the patch\u2010clamp technique, microelectrode arrays, calcium imaging, and stimulus methods, particularly optogenetic\u2010mediated light stimulation, microelectrode\u2010mediated electrical stimulation, and biochemical stimulation. 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