Isolation of subcellular fractions from a limited amount of clinical sample is imperative for the continuous advancement of biological and clinical research. For example, analyses of pure and functional mitochondria from patient samples are expected not only to expand our knowledge towards the basic biological mechanisms of mitochondrial function, but also to elucidate how mitochondria are involved in the development of diseases such as cancers, premature aging syndromes, diabetes and neurodegenerative disorders. While currently available methods are mostly laborious and not suitable for small-scale analyses, we present a novel and facile approach to isolate mitochondria from limited amounts of biological samples by centrifugal-based differential migration. Furthermore, sorting cellular organelles by their intrinsic inertia enjoys the benefits of easy operation, undemanding equipment needs and continuous batch processing. Herein, we have successfully isolated functional mitochondria from crude cell lysate of less than 100 cells, which demonstrates the possibilities of promoting this methodology for detailed analyses of subcellular organelles, particularly when small-scale clinically relevant samples are considered.