Within the research for the bachelor thesis, we evaluated the content of antioxidants in the milling fractions of three cultivars of barley. We milled the barley and sifted the flour through the first sieve (124 ?m). We milled the bigger fraction again and sifted it through sieves of sizes 124 ?m, 140 ?m and 250 ?m. Out of the flours, which represented different fractions we extracted the antioxidants and determined their content with free radicals DPPH and ABTS and with Folin-Ciocalteu reagent. The contents of antioxidants were determined from the standard curves, made with Trolox and expressed as Trolox equivalents. The results reveal that different fractions contain a vastly different amount of antioxidants. In general, the content of antioxidants in the fractions with smallest particles was the lowest. We found out that the distribution of antioxidants in the particular fractions of hulled and hull-less barley is different. For hull-less barley, the largest antioxidant content was found in the fraction with the largest particles, while the fraction with particle size between 140 ?m and 250 ?m was the richest source of antioxidants for both hulled barleys.