ScopeAs orange juice belongs to one of the most consumed juices worldwide, a human study is performed to identify urinary biomarkers for the consumption of orange juice in order to differentiate between low, medium, and high intake.Methods and ResultsThe 32 study participants abstained from citrus fruits, juices and products thereof, except for one portion of orange juice, for eight days. Throughout the study, spot urine samples are collected and quantitatively analyzed by high‐performance liquid chromatography tandem mass spectrometry (HPLC‐MS/MS) regarding their content of several potential biomarkers for orange juice intake after enzymatic treatment with β‐glucuronidase. Proline betaine is determined as a long‐term biomarker: based on its urinary excretion, orange juice consumption is traceable for at least 72 h after intake. Naringenin and hesperetin are identified as qualitative short‐term biomarkers. Synephrine sulfate also showed a fast increase and decrease in a semi‐quantitative approach. In the case of phloretin, no correlation between orange juice consumption and the urinary concentration is observed.ConclusionProline betaine is the most promising biomarker for orange juice consumption and allows to differentiate between low, medium, and high intake. Hesperetin and naringenin (as well as synephrine) are applicable as supporting biomarkers, whereas phloretin does not represent a reliable biomarker for orange juice consumption.