Nutritional markers have several advantages for epidemiologic and clinical assays, when compared to dietary data obtained by food frequency questionnaires. Few studies have assessed whether total polyphenol (TP) compounds provide a valid biomarker for TP intake. To date, there has been almost no literature describing methods to determine TP in complex matrices such as urine, which have many interfering substances. We report a rapid Folin-Ciocalteu method to determine TP in urine samples using Oasis((R)) MAX 96-well plate cartridges for solid phase extraction. These plates allow analysis of a high number of samples at the same time. We performed a prospective, randomized, crossover trial and one cross-sectional study with 60 volunteers from the PREDIMED trial, seeking to evaluate whether the TP in urine were correlated with polyphenol intake and could, therefore, be considered as a marker of intake of these compounds. The assay was optimized; the sensitivity and the polarity range of urine polyphenols were increased and the detection and quantification limits were significantly reduced. The metabolites in standards solution and urine samples were stable under the storage and handling conditions. In the clinical trial and the cross-sectional study, TP excreted in spot urine samples were positively correlated with TP intake, r=0.48, P<0.01 and r=0.257, P=0.04, respectively. The methodology described may be used to detect TP in urine samples, employing the high throughput of 96-well microtiter plates and reader. The method is fast and simple and it allows analysis of a large number of samples at the same time.