The neuromuscular junction (NMJ) is a highly specialised subcellular niche that connects motor neurons to skeletal muscle. Despite its central role in regulating movement, contraction and independence, the proteome that regulates its function remains largely undefined. Here, we developed and validated a series of TurboID-based proximity labelling constructs to study the NMJ proteome in vivo. We further validate protein localisation by optimising a laser capture microdissection protocol of skeletal muscle coupled with ultra-sensitive proteomics. These technologies were used to quantify; i) the kinetics of NMJ proteome remodeling during a time course of denervation/re-innervation induced by cryo-injury, and ii) NMJ destruction during the progression of two mouse models of Amyotrophic lateral sclerosis (ALS) motor neuron disease. The data were integrated with a novel high-throughout morphological analysis of the pre- and post-synapse to enable the correlation of individual NMJ proteins to specific cellular and subcellular phenotypes during regeneration or deterioration. These data have identified 100’s of novel proteins that are regulated at the NMJ with high spatial-temporal resolution, and we show several are also dysregulated in muscle biopsies from ALS patients. Finally, we present preliminary and ongoing pre-clinical work targeting several of these novel proteins as therapeutic targets to promote regeneration and improve muscle function in mouse models of nerve injury and ALS.