Aminoglycoside ototoxicity is common in clinical practice but reliable protective agents currently do not exist. Aminoglycoside regimens causing ototoxicity in different laboratory animals are under investigation. The assessment method used most commonly to determine auditory effects is the auditory brainstem response (ABR). Distortion product otoacoustic emissions (DPOAE) have been used less frequently. A precise recommendation on the specific method to assess peripheral auditory function before and after aminoglycoside toxicity in mice does not exist. In order to evaluate various mouse models for ototoxic injury caused by various aminoglycoside regimens, there is a need for performing preliminary tests in small cohorts before large experiments. The aim of our study was to investigate different aminoglycoside regimens that cause substantial ototoxic damage
in vivo
. Aminoglycosides are safe and produce a detectable hearing threshold shift in a small cohort of mice that can be used as a model for preliminary tests. Different ototoxic regimens were assessed by ABR and DPOAE measurements pre- and post-treatment. Further, the sensory cell loss was quantified by counting hair cells in the cochlea. It was revealed that an ototoxic regimen with kanamycin twice daily for 15 consecutive days is safe, well tolerated and produces an early significant hearing threshold shift detected by DPOAE in a small cohort of mice. The study compared ABR and DPOAE in mentioned regimens for the first time and illustrated that DPOAE is well suited for detecting hearing threshold shifts in high frequencies before ABR threshold shifts occur in accordance with predominating outer hair cell damage mainly in the basal turn of the cochlea.