The aim of the current study was to validate an automated immunoassay for cortisol quantification in the saliva of pigs. The assay had intra- and interassay coefficients of variation lower than 16%, in all cases. The limit of detection was 0.016 µg/dl, and the lower quantification limit was 0.197 µg/dl. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution and recovery tests. In addition, this assay was used to quantify cortisol in 2 models of stress: 1 in which animals were immobilized with a nose-snare and 1 in which pigs were transported for a duration of 30 min. In both cases, a significant increase (P < 0.01) in salivary cortisol was detected after the stressful situation. Overall, the assay validated in the present study could be used for the evaluation of cortisol changes in stressful situations.