Background Outcome variability in cochlear implantation (CI) is inadequately explained, with limited understanding of how the diseased cochlea impacts clinical outcomes. The recent advent of human perilymph sampling research in the CI population offers potential insights into the molecular correlates of end-stage hearing loss and their implications for hearing rehabilitation. However, expertise in perilymph sampling is currently concentrated in only a few centres globally. Methods Paediatric epidural catheters were validated through benchtop and wet lab experiments. A prospective clinical cohort trial involving 24 CI recipients was conducted, sampling perilymph during surgery. Extracochlear electrocochleography was performed before and after sampling to assess the impact on residual cochlear function. The samples underwent next-generation microRNA sequencing, and perilymph miRNA expression was correlated with clinical outcomes such as hearing preservation, speech perception performance, and impedance. Results The sampling technique proved efficient and ergonomic, with no significant impact on ECochG thresholds or amplitudes. There was no notable change in cochlear microphonic amplitude in response to a 100 dB HL stimulus (p = 0.3618) and no significant change in cochlear microphonic threshold (p = 0.8888). A trend was observed for larger cochlear microphonic amplitudes at threshold stimulus intensity (p = 0.0663). Sample yield was satisfactory for next-generation sequencing, with a mean yield of 2.34 Gb base pairs. Implications This study represents the first Australian clinical application of human perilymph sampling in CI recipients. It demonstrates a safe, effective, and transferrable surgical methodology. The expression of miRNA in perilymph may have correlations with clinical outcomes. Results of bioinformatic analysis of miRNA libraries to follow.