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Mitochondrial physiology analysis is central to cell metabolism research and understanding of many human diseases. High resolution respirometry represents an efficient approach to study mitochondrial physiology in isolated mitochondria, both permeabilized and intact cells, as well as in tissue biopsy samples. We use digitonin for controlled plasma membrane permeabilization to perform extended functional OXPHOS analysis by O2k Oxygraph in palmitate treated cells, an in vitro model of insulin resistance and lipotoxicity. Digitonin has high affinity for cholesterol, thus low digitonin concentration will completely and selectively permeabilize plasma membrane (richer in cholesterol), compared to mitochondrial and endoplasmic reticulum membranes (low in cholesterol), so the latter are affected only at higher digitonin concentrations. It is known that molecular composition of plasma membrane differs between cell types and that modification in plasma membrane lipid composition occurs in tumor cells [1]. Thus, every cell line requires previous optimization of adequate digitonin concentration, which is sufficient to permeabilize plasma membrane and which allows entrance of mitochondrial substrates and inhibitors, while keeping mitochondria intact and functional in their intracellular environment.