Mitochondrial dysfunction is discussed as a key player in the pathogenesis of type 2 diabetes mellitus (T2Dm), a highly prevalent disease rapidly developing as one of the greatest global health challenges of this century. Data however about the involvement of mitochondria, central hubs in bioenergetic processes, in the disease development are still controversial. Lipid and protein homeostasis are under intense discussion to be crucial for proper mitochondrial function. Consequently proteomics and lipidomics analyses might help to understand how molecular changes in mitochondria translate to alterations in energy transduction as observed in the healthy and metabolic diseases such as T2Dm and other related disorders. Mitochondrial lipids integrated in a tool covering proteomic and functional analyses were up to now rarely investigated, although mitochondria]. lipids might provide a possible lynchpin in the understanding of type 2 diabetes development and thereby prevention. In this chapter state-of-the-art analytical strategies, pre -analytical aspects, potential pitfalls as well as current proteomics and lipidomics-based knowledge about the pathophysiological role of mitochondria in the pathogenesis of type 2 diabetes will be discussed.