BackgroundTotal antioxidant status (TAS) is one of the most widely used oxidative stress biomarkers, but the lack of canine RI and the influence of analytical factors hinder its application in clinical practice.ObjectivesThe aims of this study were to establish canine assay‐specific RI for TAS and evaluate the sources of biological variation and the association between TAS and multiple hematologic and biochemical variables.MethodsBlood samples from 190 clinically healthy dogs were collected, encompassing pet dogs (82), police dogs (56), and shelter dogs (52). After hematologic and biochemical analysis, serum TAS was determined by means of a commercial 2,2′‐azinobis (3‐ethylbenzthiazolin‐6‐sulfonic acid) (ABTS) test. The American Society for Veterinary Clinical Pathology guidelines were followed to establish the RI, employing nonparametric methods. Univariate analysis and multivariate analysis were conducted to assess the influence of biological and analytical variables, yielding a final regression model.ResultsThe final reference population comprised 143 dogs, for which the RI was established (1.41–2.27 mmol/L). Partitioning was applied to the three study groups. The regression model revealed that police dogs had significantly higher TAS values than pet dogs. Furthermore, significant associations between four biochemical variables (albumin, globulins, cholesterol, and aspartate aminotransferase) and serum TAS were found.ConclusionsThis is the first study to establish RI for serum TAS in a large and heterogeneous canine population and provide data on its relationship with analytical variables. These findings could potentially improve the interpretation of TAS in clinical environments.