UPLC-MS/MS profiling of Cassia glauca leaves extract revealed the identification of 10 flavonoids. Kaempferol 3-O-β-D-rutinoside was isolated and studied for its cytotoxic activity. It showed high cytotoxic effects against MCF-7 (IC50 of 4.6±0.038 μg/ml) and HepG-2 (IC50 of 8.2±0.024 μg/ml) cancer cell lines, compared to the leaves extracts, their Ag nanoparticles, and doxorubicin. Moreover, Kaempferol 3-O-β-D-rutinoside exerted a synergistic cytotoxic effect with doxorubicin on MCF-7 cell lines. It was discovered as kinases and aldose reductase inhibitor while rationalizing its cytotoxic activity through molecular docking study. Thus, it is expected that the cardiotoxic effects of doxorubicin can be also decreased by using Kaempferol 3-O-β-D-rutinoside due to its aldose reductase inhibitory effect. These findings suggested that Kaempferol 3-O-β-D-rutinoside could be used in combination with chemotherapeutic drugs to increase the sensitivity to their cytotoxic activity and protect against their side effects.