The relative activities of the antioxidants Trolox, ascorbic acid, uric acid, quercetin, and rutin, and the activities of total antioxidants in serum samples were determined using a fluorometric assay based on the dye 6-carboxyfluoroscein (6C-Fl) as a fluorescent indicator; 2,2-azobis-2-amidinopropane hydrochloride (AAPH) as a peroxyl radical generator; 6-hydroxy-2,5,7,8tetramethyl-1-chroman-2-carboxylic acid (Trolox) as a calibrator; and phosphate buffer (pH 7.0) as a solvent. Incubation of 6C-Fl in 0.075 M phosphate buffer, in the presence of AAPH at 37°C, resulted in loss of its fluorescence signal at 520 nm with excitation at 495 nm. The antioxidants Trolox, ascorbic acid, and uric acid provided protection of the fluorescence of 6C-Fl, and the relative antioxidant activities, determined by the net protection area under curve technique, were found to be 1:0.4:1, respectively. Trolox and ascorbic acid were used to validate this assay. A linear correlation of the net protection value with the concentration of serum, Trolox, ascorbic acid, and uric acid was demonstrated. Quercetin and rutin were shown to have strong antioxidant activities, nearly 10 times those of vitamin C. This assay is simple, reliable, and suitable for automation to handle many samples and requires few microliters of serum samples.