BACKGROUND: Equine gastric ulcer syndrome (EGUS) is a frequent disease in horses that comprises two different entities: equine squamous gastric disease (ESGD) and equine glandular gastric disease (EGGD). This disease considerably reduces the quality of life of affected horses and can negatively affect performance. Saliva contains biomarkers, such as oxytocin, that have been used as a welfare indicator and can develop a function as a protective factor against stress-induced changes in gastric function due to its gastric antisecretory and antiulcer effects. The objective of this work was to evaluate changes in salivary oxytocin concentrations in healthy and EGUS horses. For this purpose, an immunoassay based on AlphaLISA technology was validated for the quantification of salivary oxytocin and applied in a total of 102 horses divided into 5 groups: 25 with both EGUS, 23 with only EGGD, 21 with only ESGD, 19 horses with other diseases, and 14 healthy horses. RESULTS: The analytical validation of the method showed good precision and linearity under dilution. Salivary oxytocin concentrations in healthy horses were higher compared to horses with both ESGD and EGGD and only EGGD. Salivary oxytocin concentrations in horses with only ESGD were higher compared to horses with both ESGD and EGGD and horses with only EGGD. In addition, salivary oxytocin concentrations in horses with other diseases different from ESGD were significantly increased compared to horses with both ESGD and EGGD and horses with only EGGD. CONCLUSIONS: This report validates a new assay that can measure oxytocin in saliva in horses in a precise and accurate way. The lower oxytocin values in horses with EGGD and both EGGD and ESGD than in horses with ESGD, horses with other diseases, and healthy horses could indicate a possible relation of oxytocin with this disease.