Background Oxidative stress is associated with a diverse group of liver disorders across species. Objectives Determine whether glutathione (GSH) concentration in plasma and red blood cells correlates with liver GSH concentration in dogs and evaluate whether other markers of systemic oxidative stress, plasma vitamin E and urine 8‐isoprostanes/creatinine (F2‐IsoPs/Cr) concentrations, correlate with liver GSH. Animals Thirty‐four client‐owned dogs undergoing clinically indicated liver biopsy and 15 healthy control dogs. Methods Prospective, observational cross‐sectional study. Urine and blood were collected before liver biopsy. Plasma, erythrocyte, and liver GSH were measured using high performance liquid chromatography (HPLC); vitamin E was measured by HPLC, and F2‐IsoPs/Cr was measured by gas chromatography/mass spectrometry. Results All dogs were treated at the discretion of the attending clinician (24/34 received antioxidants; 4/34 fed therapeutic liver diet), which included dogs with primary or secondary liver disease (inflammatory (n = 21), metabolic (n = 9), vascular (n = 2), and neoplastic (n = 2)). Median GSH concentrations in plasma, erythrocyte, and liver were 0.18 mg/dL (range 0.14 to 0.56 mg/dL), 56.7 mg/dL (18.3 to 79.2 mg/dL), and 181 mg/dL (39.9 to 527 mg/dL), respectively. No significant correlations were found between liver GSH and erythrocyte GSH, plasma GSH, vitamin E, or F2‐IsoPs/Cr. Dogs undergoing clinically indicated liver biopsy had significantly higher urine F2‐IsoPs/Cr than did healthy controls (5.89 vs 2.98 ng/mg; P < .0001). Conclusions and Clinical Importance Erythrocyte and plasma GSH are not indicative of liver GSH concentration in dogs. In addition, dogs undergoing clinically indicated liver biopsy have evidence of increased systemic oxidative stress compared to healthy controls.