Mitochondria are metabolic hubs, with many diseases found to have altered metabolism and mitochondrial dysfunction, such as ischaemia-reperfusion injury. A detailed understanding of the metabolic changes in different cellular pools would aid diagnosis and treatment. However, the current methods of mitochondrial isolation are too slow to provide a snapshot of purely mitochondrial metabolism, meaning that current metabolic data is only from whole cell. This project has developed and used a novel technique to rapidly isolate mitochondria from tissue by density centrifugation through silicone oil, with a view to assess the mitochondrial metabolic changes during ischaemia-reperfusion injury. This method has minimal cytosolic contamination and is completed in under 5 minutes, and mass spectroscopy analysis has shown enrichment of mitochondrial metabolites. Seahorse and Oroboros analysis have shown that the mitochondria are functional and capable of coupled respiration. Data is presented on how the method optimisation was analysed and developed. This largely reduced time frame gives the advantage over other methods to enable the study of metabolism in mitochondria.