Our results show two species for Mito-pH2 in both absorbance and fluorescence profiles. At acidic pH yellow-green fluorescence (~565 nm) from the protonated species is predominant, whereas at basic pH red fluorescence (~610 nm) from the deprotonated species is predominant. This suggests a ratiometric may be possible. Indeed, the ratio of fluorescence intensities (I610/I565) shows a strong pH dependent trend, wherein the ratio (I610/I560) shows a dramatic increase in the pH range 7-9 (Figure ) demonstrating efficacy of Mito-pH2 as a ratiometric pH-probe. Single-color fluorescence intensity-based measurements are often susceptible to factors such as concentration of the probe and instrument parameters like light intensity and detector gain, etc. However, a ratiometric approach addresses these caveats. This approach is not only independent of probe concentration and instrument parameters but is also more reliably quantitative.