Fluorescence lifetime imaging microscopy (FLIM) with fluorescent ion sensors enables the measurement of ion concentrations based on the detection of photon emission events after brief excitation with a pulsed laser source. In contrast to intensity-based imaging, it is independent of dye concentration, photobleaching, or focus drift and has thus been successfully employed for quantitative analysis of, e.g., calcium levels in different cell types and cellular microdomains. Here, we tested the suitability of CoroNaGreen for FLIM-based determination of sodium concentration ([Na+]) inside cells. In vitro measurements confirmed that fluorescence lifetimes of CoroNaGreen (CoroNaFL) increased with increasing [Na+]. Moreover, CoroNaFL was largely independent of changes in potassium concentration or viscosity. Changes in pH slightly affected FL in the acidic range (pH ≤ 5.5). For intracellular determination of [Na+], HEK293T cells were loaded with the membrane-permeable form of CoroNaGreen. Fluorescence decay curves of CoroNaGreen, derived from time-correlated single-photon counting, were approximated by a bi-exponential decay. In situ calibrations revealed a sigmoidal dependence of CoroNaFL on [Na+] between 0 and 150 mM, exhibiting an apparent Kd of ∼80 mM. Based on these calibrations, a [Na+] of 17.6 mM was determined in the cytosol. Cellular nuclei showed a significantly lower [Na+] of 13.0 mM, whereas [Na+] in perinuclear regions was significantly higher (26.5 mM). Metabolic inhibition or blocking the Na+/K+-ATPase by removal of extracellular K+ caused significant [Na+] increases in all cellular subcompartments. Using an alternative approach for data analysis (“Ratio FLIM”) increased the temporal resolution and revealed a sequential response to K+ removal, with cytosolic [Na+] increasing first, followed by the nucleus and finally the perinuclear regions. Taken together, our results show that CoroNaGreen is suitable for dynamic, FLIM-based determination of intracellular [Na+]. This approach thus represents a valuable tool for quantitative determination of [Na+] and changes thereof in different subcellular compartments.