Impaired mitochondrial function has been linked to many diseases, such as stroke, heart disease, cancer, Type II diabetes and Parkinson’s disease. Mitochondria-enriched preparations are needed for proteomic and metabolomic studies that may provide crucial insights into tissue-specific mitochondrial function and dysfunction, and answer fundamental questions of cellular energetics and oxidative stress. Extraction of mitochondria from whole tissue samples is typically performed using Potter-Elvehjem homogenizers or similar labor-intensive manual disruption methods that require extensive operator experience, and often result in damage to fragile organelles and high sample-to-sample variability. Here we describe a semi-automated method that uses a novel, gentle mechanical homogenizer (The PCT Shredder) and hydrostatic pressure to release intact mitochondria from fresh rat tissues with minimal hands-on time. Pressure Cycling Technology (PCT)-based tissue homogenization is conducted under controlled thermodynamic conditions (time, temperature and pressure) leading to more reproducible results. The quality of mitochondria preparations was characterized by electron microscopy, 2D PAGE, Western blotting and respiration assays. Our data demonstrate that mitochondria extracted by the PCT sample preparation system (PCT-SPS) are intact, functional, and exhibit a protein profile comparable to control samples isolated using a conventional Potter-Elvehjem homogenizer. The resulting mitochondria-enriched samples were also subjected to trypsin digestion followed by nanoLC-MS/MS analysis on an LTQ-Orbitrap. Proteomic profiles of mitochondria samples prepared using the novel extraction technique were compared to those extracted using a conventional manual method to demonstrate the purity of mitochondrial preparations extracted using the novel PCT-SPS method.