The epidemiological assessment of wine consumption usually has been obtained using self-reporting questionnaires. In this study, two metabolomic approaches, targeted and untargeted, were applied to 24-h urine samples from a cohort of La Rioja (Spain) (aged 52-78), comparing moderate and daily wine consumers (20 males and 13 females) without diet intervention, versus non-consumers (8 males and 35 females). Results showed that the non-targeted metabolomics approach has allowed for the annotation of sixteen compounds in 24-h urine samples from regular wine-consumers that were not detected in the urine of non-wine consumers. Additionally, the targeted metabolomics approach showed a wide range of phenol metabolites, mainly hepatic phase-II conjugates, whose concentration was significantly higher in the urine of wine consumers. As a novelty, this study focuses on discovering the main urinary biomarkers of regular wine consumption involving free-living volunteers, without dietary intervention or restrictions that might alter their regular behaviors and lifestyles.