To assess the phototriggered cleavage of proanionophore 4 and in-situ generation of anionophore 2 the 1 H NMR-based study was performed. The DMSO-d6 solution of proanionophore 4 was prepared, and the 1 H NMR spectra were recorded after irradiation by LED light (365 nm). After photo irradiation with an LED lamp (365 nm), a new peak was seen at δ = 10.25 ppm, which implies the formation of 4-formyl-3-nitrosobenzoic acid, a plausible result of the photocleavage of the proanionophore 4. The aromatic peaks of the phototriggered tether (Hb, Hc, and Hd) and benzylic proton also decrease with the increase in the duration of photoirradiation, suggesting the cleavage process (Fig. ). The successful generation of the anionophore using the NMR experiment persuaded us to study the membrane transport after phototriggered cleavage of the proanionophore 4 into anionophore 2. In this regard, we examined the ion transport across EYPC/CHOL-LUVlucigenin (prepared using 225 mM aqueous solution of NaCl) dispersed in 225 mM aqueous solution of NaH2PO4. The ion transport activities of proanionophore 4 were recorded before and after photo irradiation (Fig. ). It was perceived that the transport efficacy increased by 60%. Consequently, by both the NMR and fluorescence-based methods, the resurgence of the anionophore 2 from proanionophore 4 can be affirmed. The perturbation of chloride-phosphate ion homeostasis in the cancer cells could induce apoptosis or other cell death pathways. The MTT-based cell viability assay revealed that compound 2 induced moderate toxicity (IC50 = 20 µM) to the HeLa cells. However, the proanionophore 4 showed negligible toxicity even after treatment with 60 µM concentration. Interestingly, photo irradiation (UV 365 nm, 5 min) of proanionophore 4 promoted moderate toxicity to the HeLa cells (IC50 = 36 µM). Whereas only photo irradiation (UV 365 nm, 5 min) without the treatment of proanionophore 4 did not significantly alter the viability of the HeLa cells. Hence, the lower viability after the photo irradiation could be due to cleavage of proanionophore 4 and in-situ generation of active anionophore 2 under the cellular environment. Overall, these results successfully established that photoinduced disruption of chloride-phosphate ions homeostasis induces cancer cell death.