This study describes a simple, sensitive, and reliable method for quantifying the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a small volume (a few microliters). The method uses β-phycoerythrin (β-PE) as an indicator protein, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) as a peroxyl radical generator, and Trolox (a water-soluble vitamin E analogue) as a control standard. The ORAC value is expressed as units, where 1 ORAC unit equals the net protection produced by 1 μM Trolox. The assay is unique in that it estimates the total antioxidant capacity by driving the oxidation reaction to completion. This allows for the quantification of antioxidant protection without the need for kinetic or lag-time measurements. The method demonstrates a linear correlation between ORAC values and concentrations of serum, Trolox, vitamin C, uric acid, and bovine albumin. The coefficient of variation within a run is about 2%, and between runs is about 5%. The relative peroxyl radical absorbance capacity of Trolox, α-tocopherol, vitamin C, β-carotene, uric acid, and bilirubin is 1:1:0.92:0.84:0.52. Bovine albumin has a lower peroxyl absorbing capacity than these antioxidants. However, the serum protein fraction, containing some lipid-soluble antioxidants, represents the major contributor to the ORAC value found in whole serum. The minimum detectable amounts of vitamin C and uric acid in serum supernatant fractions are 1.5 μg and 0.59 μg, respectively, which account for about 1% of the total ORAC value of the serum supernatant fraction. The ORAC assay is also applicable for measuring α-tocopherol acid succinate, β-carotene, and bilirubin. The results show that the ORAC assay is a simple, sensitive, and reliable method for quantifying the peroxyl radical absorbing capacity of antioxidants in serum or other biological fluids. The method has been validated with human serum samples and demonstrated high repeatability and accuracy. The study also highlights the importance of serum antioxidants in protecting blood vessels from inflammatory peroxides. The ORAC assay has potential applications in evaluating the hydroxyl radical absorbing capacity of serum. The study concludes that the ORAC assay is a valuable tool for assessing antioxidant capacity in biological fluids.This study describes a simple, sensitive, and reliable method for quantifying the oxygen-radical absorbing capacity (ORAC) of antioxidants in serum using a small volume (a few microliters). The method uses β-phycoerythrin (β-PE) as an indicator protein, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) as a peroxyl radical generator, and Trolox (a water-soluble vitamin E analogue) as a control standard. The ORAC value is expressed as units, where 1 ORAC unit equals the net protection produced by 1 μM Trolox. The assay is unique in that it estimates the total antioxidant capacity by driving the oxidation reaction to completion. This allows for the quantification of antioxidant protection without the need for kinetic or lag-time measurements. The method demonstrates a linear correlation between ORAC values and concentrations of serum, Trolox, vitamin C, uric acid, and bovine albumin. The coefficient of variation within a run is about 2%, and between runs is about 5%. The relative peroxyl radical absorbance capacity of Trolox, α-tocopherol, vitamin C, β-carotene, uric acid, and bilirubin is 1:1:0.92:0.84:0.52. Bovine albumin has a lower peroxyl absorbing capacity than these antioxidants. However, the serum protein fraction, containing some lipid-soluble antioxidants, represents the major contributor to the ORAC value found in whole serum. The minimum detectable amounts of vitamin C and uric acid in serum supernatant fractions are 1.5 μg and 0.59 μg, respectively, which account for about 1% of the total ORAC value of the serum supernatant fraction. The ORAC assay is also applicable for measuring α-tocopherol acid succinate, β-carotene, and bilirubin. The results show that the ORAC assay is a simple, sensitive, and reliable method for quantifying the peroxyl radical absorbing capacity of antioxidants in serum or other biological fluids. The method has been validated with human serum samples and demonstrated high repeatability and accuracy. The study also highlights the importance of serum antioxidants in protecting blood vessels from inflammatory peroxides. The ORAC assay has potential applications in evaluating the hydroxyl radical absorbing capacity of serum. The study concludes that the ORAC assay is a valuable tool for assessing antioxidant capacity in biological fluids.