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Although there is some data from animal studies, the metabolome of inner ear fluid in humans remains unknown. Characterization of the metabolome of the perilymph would allow for better understanding of its role in auditory function and for identification of biomarkers that might allow prediction of response to therapeutics. There is a major technical challenge due to the small sample of perilymph fluid available for analysis (sub-microliter). The objectives of this study were to develop and validate a methodology for analysis of perilymph metabolome using liquid chromatography–high resolution mass spectrometry (LC-HRMS). Due to the low availability of perilymph fluid; a methodological study was first performed using low volumes (0.8 μL) of cerebrospinal fluid (CSF) and optimized the LC-HRMS parameters using targeted and non-targeted metabolomics approaches. We obtained excellent parameters of reproducibility for about 100 metabolites. This methodology was then used to analyze perilymph fluid using two complementary chromatographic supports: reverse phase (RP-C18) and hydrophilic interaction liquid chromatography (HILIC). Both methods were highly robust and showed their complementarity, thus reinforcing the interest to combine these chromatographic supports. A fingerprinting was obtained from 98 robust metabolites (analytical variability <30%), where amino acids (e.g., asparagine, valine, glutamine, alanine, etc.), carboxylic acids and derivatives (e.g., lactate, carnitine, trigonelline, creatinine, etc.) were observed as first-order signals. This work lays the foundations of a robust analytical workflow for the exploration of the perilymph metabolome dedicated to the research of biomarkers for the diagnosis/prognosis of auditory pathologies.Metabolic composition of inner ear fluid.Analytical LC-MS optimization of submicroliter sample.A targeted fingerprinting of human perilymph fluid was given.