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Quantification of cortisol concentration in hair has become a promising conservation tool for non-invasive monitoring of "stress" in wild populations, yet this method needs to be carefully validated for each species. The goals of the study were:•Immunologically validate two methods (study 1 and 2 respectively) to extract and quantify cortisol in the hair of wild Alpine marmots.•Compare the amount of cortisol extracted from hair samples using two methods i.e. cut into fine pieces (study 1) and hair samples pulverized using a ball mill (study 2).•Determine the extent to which methods in study 2 could provide individual specific hair cortisol (HC) measures when samples were taken from the same body location. Within and between individual variations in HC levels were examined from multiple hair samples from 14 subjects in study 2. We evaluated if inter-individual variations in HC levels could be explained by sex and age.At least twice the amount of cortisol was obtained per g/hair when samples were pulverized in a ball mill prior to extraction compared to when cut into pieces. Our methods demonstrated intra-individual consistency in HC at a given time point: inter-individual variation in HC was three times larger than within individual variance. Sex and age did not impact HC levels., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier B.V.)