Introduction: Mitochondria are essential in energy metabolism and cellular survival, and there is growing evidence that insulin resistance in chronic metabolic disorders, such as obesity, type 2 diabetes (T2D), and aging, is linked to mitochondrial dysfunction in skeletal muscle. Protein profiling by proteomics is a powerful tool to investigate mechanisms underlying complex disorders. However, despite significant advances in proteomics within the past two decades, the technologies have not yet been fully exploited in the field of skeletal muscle proteome. Area covered: Here, we review the currently available studies characterizing the mitochondrial proteome in human skeletal muscle in insulin-resistant conditions, such as obesity, T2D, and aging, as well as exercise-mediated changes in the mitochondrial proteome. Furthermore, we outline technical challenges and limitations and methodological aspects that should be considered when planning future large-scale proteomics studies of mitochondria from human skeletal muscle. Authors’ view: At present, most proteomic studies of skeletal muscle or isolated muscle mitochondria have demonstrated a reduced abundance of proteins in several mitochondrial biological processes in obesity, T2D, and aging, whereas the beneficial effects of exercise involve an increased content of muscle proteins involved in mitochondrial metabolism. Powerful mass-spectrometry-based proteomics now provides unprecedented opportunities to perform in-depth proteomics of muscle mitochondria, which in the near future is expected to increase our understanding of the complex molecular mechanisms underlying the link between mitochondrial dysfunction and insulin resistance in chronic metabolic disorders.