Multi-omics applications are essential tools in systems medicine and biology. Alterations in molecular fingerprints are closely related to changes in cellular and subcellular function, but a linkage of functional investigations and omics analyses is quite rare. In mitochondria, lipids, proteins and post-translational modifications thereof are directly linked to function but also to structure. However, the complementary combination of multi-omics analyses and functional investigations in mitochondrial research is challenging. Analytical chemists can implement this strategy in systems medicine projects, thereby providing comprehensive results as a base for correct data interpretation and a deeper understanding of the role of mitochondria in health and disease. In this review we will discuss state-of-the-art approaches of this multistep process focussing on analytical, but also pre-analytical aspects, like mitochondrial purification and sample quality control, as joint key for valid functional multi-omics investigations of mitochondria by complementary lipidomics, proteomics and respiratory analyses. (C) 2019 The Authors. Published by Elsevier B.V.