Metabolomics and lipidomics generally targets a huge number of intermediate and end products of cellular metabolism in body fluids, tissues, and cells etc. At present, mass spectrometry (MS) based metabolic or lipid profiling of routine biological specimens including the whole cells, tissues, plasma, serum and urine etc., can cover hundreds of metabolites or lipid species in one analysis, which has qualified deep elucidation of global metabolic and lipid networks. Mitochondria are important intracellular organelles and many critical biochemical reactions occur here, they provide building block for new cells, control redox balance, participate in apoptosis and behave as a signalling platform. Evidence suggests high prevalence of mitochondrial dysfunction occurs in a variety of cancers and other diseases, thus there is an urgent demand for investigating and clarifying mitochondrial metabolic and lipid alterations induced by diseases. Nevertheless, mitochondria contribute a small fraction to cellular contents, profiling of whole cell is probably unsuitable for monitoring alterations in mitochondria. Therefore, metabolomics and lipidomics analyses specially for mitochondria are necessary to understand disturbed metabolic and lipid pathways induced by environment and diseases. However, methods for comprehensively profiling metabolites and lipids in mitochondria have been limited at present. This review summarizes the current states and progress of MS-based mitochondrial metabolomics and lipidomics study. Details of mitochondrial isolation procedure, analytical methods and their applications are described. The challenges and opportunities are also given.