Several clinical and epidemiological studies have shown that moderate wine consumption may exert a protective effect against oxidative stress involved in several diseases, such as cardiovascular and neurodegenerative disorders. However, the epidemiological assessment of wine consumption has usually been obtained using self-reported questionnaires containing less reliable information for assessing total intake than nutritional biomarkers. A reliable biomarker for wine consumption is, therefore, needed. To validate urinary resveratrol metabolites (RMs) as a biomarker of wine consumption in a large cohort of free-living subjects, 1000 consecutive subjects entering a substudy of the PREDIMED trial (Prevención con Dieta Mediterránea) were evaluated. Data were collected in a validated semiquantitative food frequency questionnaire. RMs were measured in morning urine by LC-MS/MS. Urinary RM values correlated directly with reported daily amounts of wine consumed (r=0.895; p<0.001). One drink of wine per week can be detected. Using a cut-off of 411.4 nmol/g creatinine, the measurement of urinary RMs could discriminate wine consumers from non-wine consumers with a sensitivity of 93.3% (95% confidence interval (CI) 91.5-94.7%) and a specificity of 92.1% (CI 90.2-93.7%). Urinary RMs fulfill the criteria to be considered as a nutritional biomarker of wine consumption in a large sample of free-living subjects. This biomarker would provide an additional tool for investigating more precisely the relationship between wine consumption and health benefits.