Intracellular pH is a key factor in cell homeostasis, regulated within specific compartments, and changes in pH can result from or affect biochemical pathways. This study explores a yellow fluorescent protein EYFP-G65T as a core for a time-resolved pH-indicator. Among the tested designs-a circular permutant, a chimeric SypHer3s-like construct, and an unmodified protein-the unmodified EYFP-G65T performed best for live-cell imaging. Upon two-photon excitation, purified EYFP-G65T exhibited a 4.5-fold increase in mean fluorescence lifetime across pH 5.5-7 and a 7-fold change in its major component's lifetime from pH 6.5-8. Using this indicator, we measured pH values ranging from 6 to 8 in various organelles, and mapped pH shifts in mitochondria and the Golgi apparatus in response to stimuli.