This work presents an experimental verification of the universal model of dissipative metabolism using the fluorescent probe MitoThermo Yellow (MTY) to record local temperature in mitochondria. Previously, the author calibrated the model on human ATP synthase and obtained numerical values of key parameters, including the reinvestment efficiency α_total = 0.15–0.25. Based on the analysis of the physicochemical properties of MTY and its interaction with components of the inner mitochondrial membrane, it has been established that MTY is a cationic lipophilic fluorophore belonging to the xanthene family. It accumulates in mitochondria due to the membrane potential (ΔΨm) and binds to components of the inner membrane. The spatial localization of the probe coincides with the region of generation of the metabolic power quantum P predicted by the model. The temperature sensitivity of MTY is due to an increase in the rate of non-radiative relaxation of the fluorophore with increasing temperature of the microenvironment. The specificity of the signal has been experimentally confirmed: it is insensitive to changes in pH, viscosity, Ca2+ concentration, and reactive oxygen species in the physiological range. Inhibition of the respiratory chain leads to a drop in the MTY signal. The greatest effect is caused by oligomycin (an ATP synthase inhibitor), reducing the estimated local temperature by 17–19°C, which allows estimating the absolute temperature of active mitochondria at no less than 54°C. The drop in signal upon inhibition of other complexes quantitatively agrees with the fraction of dissipated energy α_total = 0.15–0.25 obtained from model calibration. Independent confirmation of the model is provided by data obtained with the genetically encoded indicator mito-gTEMP, which quantitatively agree with MTY measurements in several cell lines (HEK293T, U2OS, iMEF). Cell-dependent variability in MTY binding to inner membrane components has been identified, which requires caution in interpreting the signal in some cell types and indicates the need to account for the consolidation parameter k in the model. The obtained results allow interpreting the MTY signal as a direct experimental measure of the metabolic power quantum P and confirm the fundamental proposition of the model about the inevitability of local energy dissipation in working mitochondria as an inevitable consequence of the biochemistry of oxidative phosphorylation.