In finfish aquaculture, dietary antioxidants have been shown to improve indicators of general fish health and to inhibit the oxidative deterioration of polyunsaturated fatty acids. To facilitate the characterization of novel antioxidants or antioxidant mixtures, we developed assays for antioxidant activity in a fish cell line. We used 2′,7′- dichlorodihydrofluorescein diacetate (H2DCFDA) to determine the protective effects of a panel of representative antioxidant compounds against the formation of reactive oxygen species (ROS) under conditions that promote oxidative stress, whereas protective effects against lipid peroxidation were measured using the thiobarbituric acid reactive substances (TBARS) assay and a novel implementation of 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a- diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591). We found that the highly hydrophilic antioxidant, sodium ascorbate, inhibited H2DCFDA oxidation but had no effect on lipid peroxidation, whereas the highly hydrophobic antioxidant, α-tocopherol, potently inhibited lipid peroxidation but did not prevent H2DCFDA oxidation. The data suggest that a single assay is not sufficient for estimating antioxidant activity in cultured fish cells.