Skeletal muscle mass and function progressively decline with ageing. This decline is known as sarcopenia and is a leading cause of mortality in older individuals. Loss of proteostasis is a common feature of sarcopenia, however the molecular mechanisms involved are poorly understood. Protein ubiquitylation is a key signal for maintaining cellular proteostasis. Therefore, obtaining a comprehensive understanding of protein ubiquitylation events in ageing muscle will provide insights into the molecular mechanisms involved in muscle proteostasis. We have developed a mass spectrometry-based workflow that allows for large-scale analysis of protein ubiquitylation in skeletal muscle. To improve the dynamic range of ubiquitylated protein detection, we included a fractionation process to separate myofibrillar and sarcoplasmic enriched proteins. We employed a cost-effective clean up method called SP4 to remove contaminants and deliver high protein recovery prior to trypsin digest. Finally, we used antibodies to selectively enrich ubiquitylated peptides for mass spectrometry analysis. Human skeletal mixed muscle (n=3) was used to develop this workflow. Label free quantification paired with t-test statistical analysis was used to determine fraction enriched ubiquitylated proteins. We were able to detect 4,591 unique ubiquitylated peptides and 971 unique ubiquitylated proteins. Of these proteins, 710 (73%) were significantly enriched into either muscle fraction. Over 70% of the fraction enriched ubiquitylated proteins were identified in the sarcoplasmic fraction, including heat shock proteins which are important for protein folding. Currently, we are applying this workflow to investigate changes in protein ubiquitylation between young (6 month) and old (22 month) C57BL/6 male mouse gastrocnemius muscle (n=3). Western blot analysis has shown higher abundance of protein ubiquitylation in aged, compared to young muscle. We will run these samples using our mass spectrometry workflow coupled with isobaric labels to obtain a quantitative dataset to determine which proteins undergo altered ubiquitylation in aged muscle. We believe that our new methodology will improve our understanding of the molecular mechanisms contributing to the age-related decline in muscle proteostasis. This information is critical for developing pharmacological interventions aimed at restoring muscle health and achieving healthy ageing. Ethical approval for human samples was obtained through the East Midlands – Derby Research Ethics Committee (18/EM/0004), conformed to the requirements of Research Governance at the University of Birmingham and was conducted in accordance with the Declaration of Helsinki. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Iowa.