ObjectivesOral or intratympanic corticosteroids are commonly used to treat sudden sensorineural hearing loss (SSHL), tinnitus, and Meniere disease. Direct intracochlear delivery has been proposed to overcome the variability in bioavailability and efficacy of systemic or middle ear delivery. In this study, we aim to characterize the physiologic consequences of microneedle‐mediated direct intracochlear injection of dexamethasone through the round window membrane (RWM).MethodsIn Hartley guinea pigs (n = 5), a post‐auricular incision followed by bullostomy was made to access the round window membrane. Using 100 μm diameter hollow microneedles, 1.0 μl of 10 mg/ml dexamethasone was injected through the RWM over 1 min. Compound action potential (CAP) and distortion product otoacoustic action emissions (DPOAE) were measured before perforation, at 1 h, and at 5 h following injection. CAP hearing thresholds were measured from 0.5 to 40 kHz, and DPOAE f2 frequencies ranged from 1.0 and 32 kHz. Repeated measures ANOVA followed by pairwise t‐tests were used for statistical analysis.ResultsANOVA identified significant CAP threshold shifts at four frequencies (4, 16, 36, and 40 kHz) and differences in DPOAE at 1 frequency (6 kHz). Paired t‐tests revealed differences between the pre‐perforation and 1 h time point. By 5 h post injection, both CAP hearing thresholds and DPOAE recover and are not significantly different from baseline thresholds.ConclusionDirect intracochlear delivery of dexamethasone via microneedles results in temporary shifts in hearing thresholds that resolve by 5 hours, thus supporting microneedle technology for the treatment of inner ear disorders.Level of EvidenceN/a Laryngoscope, 2023