A novel high-throughput assay for measuring antioxidant capacity against superoxide anion has been developed and validated. In this assay, hydroethidine (HE), a fluorescent probe, is oxidized by superoxide anion generated by xanthine and xanthine oxidase and increases its fluorescence intensity. Therefore, the inhibition of loss of HE's fluorescence intensity in the presence of antioxidant is an index of antioxidant capacity. The result is expressed as superoxide dismutase (SOD) equivalent. Unlike other probes, such as tetrazolium and lucigenin, one major advantage of this assay is that the use of HE is not prone to artifact. The method was rigorously validated through linearity, precision, accuracy, and ruggedness. The linear range, limit of quantitation (LOQ), and limit of detection (LOD) are 0.22-3.75 units/mL, 0.30 unit/mL, and 0.10 unit/mL, respectively. A wide variety of phenolic compounds and fruit extracts were analyzed.