Coffee brew is an integral part of the individual diet worldwide. Roasted coffee contains numerous bioactive substances whose significance for health is investigated in nutritional studies. Food biomarkers are recommended to correlate coffee consumption and health effects in the most unbiased way possible. Metabolites of atractyligenin derivatives from roasted coffee have been suggested as candidate analytes indicating coffee consumption. UHPLC-MS/MS analysis revealed that atractyligenin (1), 2-O-β-D-glucosylatractyligenin and 3'-O-β-D-glucosyl-2'-O-isovaleryl-2-O-β-D-glucosylatractyligenin were extracted into coffee brew. Their concentrations in filtered and unfiltered coffee did not differ significantly, suggesting independence from the preparation method. In a coffee intervention study (n=12, female/male 6/6), atractyligenin metabolites were not detectable in plasma after three days of coffee abstinence. After coffee, atractyligenin (1) and atractyligenin-19-O-D-glucuronide (M1) were the quantitatively dominant atractyligenin metabolites in plasma and showed two peaks each after 0.5 and 10 h, respectively. Half-lives after the first cmax in plasma were ∼0.31 h. 1 and M1 were detectable in plasma, indicating coffee consumption for up to 24 h after one serving. Within 10 h, ∼13.7% of the atractyligenin glycosides supplied by coffee brew were excreted in urine as metabolites 1 and M1. Metabolites 2β-hydroxy-15-oxoatractylan-4α-carboxy-19-O-β-d-glucuronide (M2) and 2β-hydroxy-15-oxoatractylan-4α-carboxylic acid-2-O-β-d-glucuronide (M3) were detected in only some samples and appeared unreliable as indicators for coffee consumption. No concentration differences between female and male study participants were observed in plasma and urine. In conclusion atractyligenin and its 19-O-β-D-glucuronide are promising markers of Arabica coffee consumption in plasma and urine for both men and women, independent of the brewing method.