Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O₂ ^•−) and particularly hydrogen peroxide (H₂O₂). Since H₂O₂ plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H₂O₂ production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry. Fundamental instrumental and methodological parameters were optimized for analysis of the effects of various substrate, uncoupler, and inhibitor titrations (SUIT) on respiration versus H₂O₂ production. The sensitivity of the H₂O₂ assay was strongly influenced by compounds contained in different mitochondrial respiration media, which also exerted significant effects on chemical background fluorescence changes. Near linearity of the fluorescence signal was restricted to narrow ranges of accumulating resorufin concentrations independent of the nature of mitochondrial respiration media. Finally, we show an application example using isolated mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H₂O₂ production in SUIT protocols.