Hemoglobin (Hb) toxicity is a known contributor to acute kidney injury (AKI), particularly under hemolytic conditions where cell-free Hb is present in circulation. When the endogenous Hb scavenger protein haptoglobin becomes saturated with cognate ligand Hb, excess unbound cell-free Hb extravasates into tissues, including the kidneys, leading to oxidative stress, inflammation, and impaired renal function. Although sex-based differences in AKI susceptibility have been observed, the protective role of hormones in modulating the severity of Hb-induced kidney injury remains unclear. In this study, we evaluated the impact of biological sex, specifically estrous cycle status, on renal responses to acute Hb exposure. Glomerular filtration rate (GFR) was measured noninvasively using the Medibeacon transdermal device that detects clearance of a fluorescent tracer (FITC-Sinistrin). Across all groups, the GFR declined at 2 h post-exposure to Hb; however, only females in estrous fully recovered by 24 h. Males and non-estrous females showed sustained reductions in GFR, suggesting impaired renal recovery. Injury biomarkers for kidney, liver, urine, and heart, including KIM-1, bilirubin, creatinine, troponin, and others, showed significant improvement in females during estrous compared to males and non-estrous females. All groups exhibited some degree of toxicity from the Hb, as demonstrated by elevated levels of markers compared to the control group. Nonetheless, these findings suggest that the hormonal levels related to the estrous cycle protect against cell-free Hb-induced acute renal and cardiac injury, potentially through modulation of inflammatory signaling pathways. Understanding sex- and hormone-dependent responses to Hb toxicity is critical for developing targeted therapies.