Ferroptotic cell death is significantly regulated by glutathione (GSH), an important antioxidant. This work describes the development of a novel, small fluorescent probe capable of detecting GSH levels and providing an understanding of the inhibitory effect of rutin, a naturally occurring flavonoid, during ferroptosis. The probe N-GSH exhibited outstanding sensitivity and selectivity towards GSH, as witnessed by the significant increase in fluorescence observed after GSH binding. Additionally, the probe N-GSH determined the possible inhibitory effect of rutin and the role of GSH in ferroptosis. Using fluorescence imaging, the probe N-GSH revealed that GSH levels reduced significantly during ferroptosis induction, demonstrating its vital role in the process. Furthermore, by significantly preserving GSH levels, rutin treatment prevented ferroptosis. Moreover, in comparison to additional typical ferroptosis inhibitors, such as dihydrolipoic acid (DHLA) and vitamin E (VE), it was hypothesized that rutin's inhibitory effect on ferroptosis was achieved through the synergistic effect of its antioxidant and chelate properties on Fe(II) ions.