Hydrogen peroxide (H₂O₂) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H₂O₂ in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H₂O₂ levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H₂O₂ to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H₂O₂ that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H₂O₂ with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H₂O₂ correlates with aging, it may not be causative.