Cystic fibrosis patients are often adminstered tobramycin to treat pulmonary infections. Unfortunately, a common side effect is hearing loss, which can fluctuate. Ebselen has known anti-inflammatory properties and could reduce the incidence and severity of tobramycin-induced hearing loss.In vitro: neonatal cochlear cultures were treated with tobramycin or cotreated with tobramycin and ebselen for 3 days. In vivo: adult mice were injected with tobramycin or tobramycin and ebselen for 14 days. ABRs were collected in a repeated measures design until 56 days after treatments. ABR threshold shifts were analyzed and a novel cochleotoxic criteria applied to determine the incidence of ototoxicity. Cochlear immunohistology was analyzed for IHC and OHC loss.Tobramycin leads to significant IHC and OHC loss in cochlear explant cultures. Ebselen co-treatment at 1:20 concentrations resulted in significant otoprotection. Tobramycin leads to significant ABR threshold shifts that are ameliorated by ebselen co-treatment. Hearing loss did not correlate with significant IHC or OHC loss.This mouse model of tobramycin-induced ototoxicity is clinically relevant in that it results in an incidence and severity of hearing loss recently documented in clinic. The in vitro experiments show that tobramycin kills hair cells and that ebselen co-treatment can attenuate this ototoxicity. The in vivo model shows tobramycin-induced hearing loss is ameliorated by ebselen co-treatment, but this is not explained by concomitant hair cell loss. These preclinical data support the testing of ebselen in CF patients receiving tobramycin treatment.