Mitochondria,　pivotal　organelles　in　cellular　metabolism　and　energy　production,　have　emerged　as　critical　players　in　the　pathogenesis　of　cancer.　This　review　outlines　the　progress　in　mitochondrial　profiling　through　mass　spectrometry-based　metabolomics　and　its　applications　in　cancer　research.　We　provide　unprecedented　insights　into　the　mitochondrial　metabolic　rewiring　that　fuels　tumorigenesis,　metastasis,　and　therapeutic　resistance.　The　purpose　of　this　review　is　to　provide　a　comprehensive　guide　for　the　implementation　of　mitochondrial　metabolomics,　integrating　advanced　methodologies-including　isolation,　detection,　and　data　integration-with　insights　into　cancer-specific　metabolic　rewiring.　We　first　summarize　current　methodologies　for　mitochondrial　sample　collection　and　pretreatment.　Furthermore,　we　then　discuss　the　recent　advancements　in　mass　spectrometry-based　methodologies　that　facilitate　the　detailed　profiling　of　mitochondrial　metabolites,　unveiling　significant　metabolic　reprogramming　associated　with　tumorigenesis.　We　emphasize　how　recent　technological　advancements　have　addressed　longstanding　challenges　in　the　field　and　explore　the　role　of　mitochondrial　metabolism-driven　cancer　development　and　progression　for　novel　drug　discovery　and　translational　research　applications　in　cancer.　Collectively,　this　review　delineates　emerging　opportunities　for　therapeutic　discovery　and　aims　to　establish　a　foundation　for　future　investigations　into　the　therapeutic　modulation　of　mitochondrial　pathways　in　cancer,　thereby　paving　the　way　for　innovative　diagnostic　and　therapeutic　approaches　targeting　mitochondrial　pathways.