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The aggregates of microtubule-associated protein Tau are the major hallmark of Alzheimer’sdisease. Tau aggregates accumulate intracellularly thus leading to generation of neuronal toxicity.Numerous approaches have been targeted against Tau protein aggregation, which include application of synthetic and natural compounds. Toluidine blue is a basic dye of phenothiazine family, which irradiation with 630 nm light converted to photo-excited form leading to generation of singlet oxygen species. In present work we studied the potency of Toluidine blue and photo-excited Toluidine blue against Tau aggregation. Biochemical and biophysical analysis using ThSfluorescence, SDS-PAGE, CD spectroscopy and electron microscopy suggested that Toluidine blueinhibits the aggregation of Tau in-vitro. The Photo-excited toluidine blue potentially dissolved the matured Tau fibrils, which was indicating disaggregation property of Toluidine blue. The cell biology studies including cytotoxicity assays, ROS production assays suggested Toluidine blue to be a biocompatible dye as reduced ROS levels and cytotoxicity was observed after exposure of Toluidine blue on Tau stressed cells. The photo-excited Toluidine blue modulates the cytoskeleton network in cells, which was supported by immunofluorescence studies of neuronal cells. The studies in UAS Tau E14 transgenic Drosophila model suggested that photo-excited Toluidine blue was potent to restore the survival and memory deficit of Drosophila. The overall findings of our studies suggests that Toluidine blue to be a potent molecule in rescuing the Tau-mediated pathology by inhibiting its aggregation …