Although chloride plays an important role in many cellular processes, there is a lack of data about intracellular chloride concentrations [Cl−]i, particularly due to technical problems. To overcome that, in this study fluorescence lifetime imaging microscopy in the time-domain by using time-correlated single-photon counting was combined with two-photon excitation (2P-FLIM). This 2P-FLIM setup has been successfully used with the Cl−-sensitive fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxy-quinolinium bromide (MQAE) in order to measure [Cl−]i in cockroach salivary glands, a well-established model system for studying epithelial ion transport processes. MQAE was well suitable for two-photon excitation, when loaded into cells, and displayed a sufficient dynamic range of its fluorescence decay time changes in response to variation of [Cl−]i according to the Stern–Volmer relationship. On this basis a uniform [Cl−]i in the range of 42–80 mM with a mean value of 59 mM ± 1 mM was found in resting cockroach salivary ducts, indicating active Cl− accumulation. However, exposure to Cl−-free saline caused only a moderate [Cl−]i drop to 48 mM ± 4 mM, suggesting a relatively low basolateral Cl− permeability in ducts, at least under resting conditions. Additionally, bath application of the biogenic amine dopamine, known to stimulate the saliva modification in the ducts, caused no significant [Cl−]i changes. These results suggest a more complex scenario of [Cl−]i homeostasis in cockroach salivary ducts. In conclusion, 2P-FLIM seems to be a suitable technique for quantitative [Cl−]i measurements in many biological systems.