The aim of the present report was to develop and validate new automated spectrophotometric assays for measurement of total antioxidant capacity (TAC), thiols, and advanced oxidation protein products (AOPP) in mussel gills, digestive gland and hemolymph samples, and to evaluate their possible utility in biomonitoring programs. Total antioxidant capacity (TAC) was measured by different methods: trolox equivalent antioxidant capacity (TEAC1 and TEAC2), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing ability of plasma (FRAP). The assays were precise, accurate and provided low limits of detection. When oxidative stress was promoted by inducing hypoxia and the behaviour of these biomarkers between hypoxic and controls mussels were compared, statistically significant differences were observed in all biomarkers and tissues evaluated. The results of the present study demonstrated that these biomarkers, not previously studied in mussels, show a potential use as biomarkers of oxidative stress in this species since they were validated and showed changes under a state of oxidative stress.