Triphenylphosphonium hydroethidine (TPP-HE) is a membrane-permeable probe that reacts with superoxide and forms hydroxytriphenylphosphonium ethidium (OH-TPP-E+), a fluorescent product that has been previously used in qualitative measurements of superoxide production. In order to develop quantitative methods to measure superoxide, it is necessary to take into consideration the principles that drive TPP-HE accumulation into various subcellular compartments. In the mitochondria matrix, TPP-HE accumulation depends on the mitochondrial membrane potential, which varies from cell to cell. Here we address this issue by including rhodamine 123 (R123) as an internal mitochondrial membrane potential calibrant in chemical cytometry experiments. After loading with TPP-HE and R123, a single cell is lysed within a separation capillary and its contents are separated and detected by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF). Using theoretical arguments, we show that the ratio [OH-TPP-E +]/[R123] is adequate to obtain a relative quantitation of mitochondrial matrix superoxide levels for each analyzed cell. We applied this method to single skeletal muscle myoblasts and determined that the steady state superoxide levels in the mitochondrial matrix is ∼(0.29±0.10) × 10-12 M. The development of this quantitative method is a critical step toward establishing the importance of reactive oxygen species in biological systems, including those relevant to aging and disease.