To further validate the working mechanism behind the pH sensitivity of Mito-pH2, we performed quantum chemical calculations using the Density Functional Theory (DFT) and 1 H-NMR studies. DFT calculations in an aqueous medium using the Polarizable Continuum Model (PCM) solvation model were performed to account for the observed photophysical changes in different pH conditions ). The ground state (S0 state) geometries of the protonated and deprotonated form of Mito-pH2 (Figure ) were optimised using B3LYP/6-311G(d, P) basis sets. Time depemdent-DFT calculations were carried out with these optimised geometries to check the nature of ground-state transitions (absorbance), and finally the Frontier Molecular Orbitals (FMO) were derived. Mito-pH2 in the ground state shows the major absorbance corresponds to HOMO-LUMO with an oscillator strength of f = 0.847. In contrast, the deprotonated form showed strong absorbance due to the HOMO-LUMO transition with an oscillator strength of f = 1.873, in line with the stronger absorbance observed in the basic pH in the pH titration.