Insulin regulates skeletal muscle protein degradation but the types of proteins being degraded in vivo remains to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as Low Molecular Weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin treated streptozotocin (STZ) diabetic mice for 6 and 96 hours and for 8 hours in Type-I Diabetic humans (T1D) for comparison to insulin treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed ~80% of proteins were consistently detected. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation including those involved in mitochondrial function, proteome homeostasis, nDNA support and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit (+0.5 fold, p=0.007) and cytochrome c oxidase subunit 6 (+0.305 fold, p=0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increased degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis especially of mitochondria.