Researchers have identified several methods for treating acute myocardial infarction (AMI) patients affected by ischemia and reperfusion injury. Some of these therapies include thrombolysis, balloon angioplasty, and coronary arterial bypass graft (CAGB). This lab has previously demonstrated that transplantation of mitochondria into the ischemic zone of a rabbit heart during reperfusion significantly improved recovery as compared to current techniques. In order for this therapy to be translated into the clinic a rapid isolation method for producing highly pure and functional mitochondria will be required. Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 minutes to complete. Herein, a method for rapid isolation of mitochondria from mammalian tissue biopsies is described. In this protocol, manual homogenization is replaced with the tissue dissociator's standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters which eliminates repetitive centrifugation steps. Mitochondrial isolation time is less than 30 minutes compared to 60-100 minutes using alternative methods. This isolation protocol yields approximately 2 x 10^10 viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.