UFMylation is a ubiquitin-like protein post-translational modification of Ubiquitin Fold Modifier 1 (UFM1) applied to substrate proteins. UFMylation is important for development and regulates several cellular processes such as protein quality control. Here, we describe the development of an antibody-based enrichment approach to immunoprecipitate in vivo remnant UFMylated peptides and identification by mass spectrometry. We used this approach to identify >200 UFMylation sites from various mouse tissues revealing extensive modification in skeletal muscle. In vivo knockdown of the UFMylation E2 ligase, UFC1 followed by enrichment and analysis of remnant UFMylated peptides quantified concomitant down-regulation and validation of a subset of modification sites, particularly myosin UFMylation. Furthermore, we show that UFMylation is increased in skeletal muscle biopsies from people living with Amyotrophic Lateral Sclerosis (plwALS). Quantification of UFMylation sites in these participant biopsies with multiplexed isotopic labeling reveal prominent increases in myosin UFMylation. Finally, in silico modelling suggest UFMylation of myosin directly adjacent to the ATP-binding site may regulate stability and/or function. Our data suggest that although UFMylation is not as widespread as ubiquitylation, its in vivo status is more complex than previously thought.