Mitochondria are highly heterogeneous organelles that likely have unique isoelectric points (pI), which are related to their surface compositions and could be exploited in their purification and isolation. Previous methods to determine pI of mitochondria report an average pI. This article is the first report of the determination of the isoelectric points of individual mitochondria by capillary isoelectric focusing (cIEF). In this method, mitochondria labeled with the mitochondrial-specific probe 10-N-nonyl acridine orange (NAO) are injected into a fused-silica capillary in a solution of carrier ampholytes at physiological pH and osmolarity, where they are focused then chemically mobilized and detected by laser-induced fluorescence (LIF). Fluorescein-derived pI markers are used as internal standards to assign a pI value to each individually detected mitochondrial event, and a mitochondrial pI distribution is determined. This method provides reproducible distributions of individual mitochondrial pI, accurate determination of the pI of individual mitochondria by the use of internal standards, and resolution of 0.03 pH units between individual mitochondria. This method could also be applied to investigate or design separations of organelle subtypes (e.g., subsarcolemmal and interfibrillar skeletal muscle mitochondria) and to determine the pIs of other biological or nonbiological particles.