The quantification of cortisol in hair samples is a relatively recent method that has been increasingly used for the evaluation of long-term cortisol secretion and chronic stress levels, in both humans and non-human animals. The use of hair presents many advantages in relation to the use of other biological matrices, e.g. a very low invasiveness of the sample collection. Besides, cortisol analysis in hair samples provides measurements over a long period of time, which can be used as a chronic stress indicator. Nevertheless, the physiology of cortisol in hair and the evaluation of reliable methods for its quantification in hair samples need to be further investigated. The aim of this study was to perform a partial analytical validation for the quantification of cortisol in domestic dog hair samples using a commercially available high sensitivity salivary cortisol enzyme immunoassay kit. Results on both precision and linearity tests were overall favorable, supporting that the kit can be used to reliably measure hair cortisol concentrations in dogs.