Detection and quantification of the highly reactive and short-lived superoxide (O•2-) can be challenging. Here, we present a new mass spectrometry (MS)-based method to detect and quantify O•2- using three fluorogenic hydroethidine probes: hydroethidine (HE), mito-hydroethidine (mito-HE), and hydropropidine (HPr+), which measure cytosolic, mitochondrial, and extracellular O•2-, respectively. The probes and their oxidation products were simultaneously quantified by applying multiple reaction monitoring (MRM) with MS that allowed the specific measurement of reactive oxygen species (ROS) distribution within the cell. The advantage of this liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is that coeluting compounds can be precisely distinguished using specific precursor and fragment masses. This method overcomes limitations from spectral overlap of O•2--specific and nonspecific products in fluorescence spectra or the low specificity associated with chromatography-based approaches. However, our experiments showed that these HE probes can be prone to autoxidation during incubation at 37°C in Hank's solution. Cell treatments with strong oxidants did not significantly increase levels of the O•2- radical. Thus, subtle changes in ROS levels in cell culture experiments might not be quantifiable. Our findings raise the question of whether HE-based probes can be used for the reliable detection of O•2- radicals in cell culture. Antioxid. Redox Signal. 00, 000-000.