Abstract The oxygen radical absorbance capacity (ORAC–FL) assay, a useful measure of the antioxidant capacity (AC) reported for some biological samples, supplements, and food samples, was standardized and validated for measuring AC of human milk. Limits of linearity, precision and accuracy of the ORAC–FL assay were made by constructing a Trolox Calibrator which included the addition of human milk as the sample matrix. AC assay results indicated excellent linearity ( R 2 = 0.990 ± 0.005), precision (2.2%) and accuracy on recovery (94.8 ± 3.2%) over a wide range of Trolox concentrations. To validate the assay further, mature milk samples were collected from 100 lactating mothers in Vancouver and Winnipeg, and were measured for vitamin E isomers by HPLC and AC using the standardized ORAC–FL assay. Alpha-tocopherol (α-Toc) was the major vitamin E isomer detected in human milk, established using both ultra-violet and fluorescent detection methods. Milk α-Toc concentrations were found to correlate significantly ( P R = 0.439, n = 60) and Winnipeg ( R = 0.408, n = 40). Although milk is a complex matrix with multiple components possessing potential antioxidant activity, our results indicated that the ORAC–FL assay is a very useful indicator for assessing the antioxidant capacity of human milk.