OBJECTIVE: To determine if oxidative stress induces phosphatidylserine (PS) externalization in canine erythrocytes and if exposure to antioxidants prevents such changes. METHODS: This was an in vitro, experimental study using 5 healthy, adult, purpose-bred research Beagles. Fresh EDTA-anticoagulated blood samples were collected from each dog, and erythrocytes were harvested. For objective 1, erythrocytes were exposed to the pro-oxidant agents tert-butyl hydroperoxide (TBHP) at 2, 3, or 4 mM or 2,2'-azobis(2-amidinopropane) dihydrochloride at 30, 40, or 50 mM. For objective 2, erythrocytes were exposed to 3 mM TBHP and the antioxidant N-acetylcysteine-amide (NACA) at various concentrations (0, 1, or 3 mM). Erythrocytes incubated with benzoylbenzoyl-ATP were used as positive control, whereas erythrocytes incubated with sodium chloride medium with 0.1% bovine serum albumin, DMSO, and NACA were used as negative controls. Erythrocytes were stained with allophycocyanin-conjugated Annexin V, and PS externalization was assessed by flow cytometry. The degree of PS externalization of each sample was recorded as median fluorescence intensity and percentage of PS positivity. RESULTS: TBHP at 3 and 4 mM caused increased PS externalization in canine erythrocytes. 2,2'-Azobis(2-amidinopropane) dihydrochloride at all concentrations caused increased PS externalization. N-acetylcysteine-amide at all concentrations prevented significant PS externalization measured by median fluorescence intensity and percentage of PS positivity from erythrocytes exposed to TBHP. CONCLUSIONS: Oxidative stress causes PS externalization in canine erythrocytes, and NACA ameliorates this effect. CLINICAL RELEVANCE: Future studies are needed to determine if increased PS externalization in erythrocytes occurs in dogs with immune-mediated hemolytic anemia and its role in promoting thromboembolism.