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Background: Cortisone is derived from cortisol through the action of the enzyme 11β-hydroxysteroid dehydrogenase type II, and it has gained importance in recent years as a biomarker of stress. This study aimed to develop and validate an assay for the measurement of cortisone in pig saliva and evaluate whether its concentration varies in stressful situations. For this purpose, a specific immunoassay was developed and validated analytically, and a study was performed to evaluate whether cortisone concentrations in saliva can vary under heat stress conditions., Results: The assay proved to be accurate, reliable, and sensitive for the measurement of cortisone in pig saliva. The limit of detection of the assay was set at 0.006 ng/ml, and the lower limit of quantification was 0.023 ng/ml. It also correlated significantly with the results obtained by LC‒MS/MS (P = 0.003; r = 0.64). In addition, the cortisone concentration in animals subjected to prolonged heat stress decreased significantly 15 days after treatment (P < 0.0001)., Conclusions: According to these results, cortisone measured by this assay could be used as a tool for the non-invasive evaluation of thermal stress in pig saliva., (© 2024. The Author(s).)