Grape-derived polyphenols are considered to be one of the most promising ingredients for functional foods due to their health-promoting activities. We applied a HPLC-MS-based untargeted metabolomic approach in order to evaluate the impact of a functional food based on grape skin polyphenols on the urinary metabolome of healthy subjects. Thirty-one volunteers participated in two dietary crossover randomized intervention studies: with a single-dose intake (187 mL) and with a 15-day sustained consumption (twice per day, 187 mL per day in total) of a functional beverage (FB). Postprandial (4-hour) and 24-hour urine samples collected after acute consumption and on the last day of sustained FB consumption, respectively, were analysed using an untargeted HPLC-qTOF-MS approach. Multivariate modelling with subsequent application of an S-plot revealed differential mass features related to acute and prolonged consumption of FB. More than half of the mass features were shared between the two types of samples, among which several phase II metabolites of grape-derived polyphenols were identified at confidence level II. Prolonged consumption of FB was specifically reflected in urine metabolome by the presence of first-stage microbial metabolites of flavanols: hydroxyvaleric acid and hydroxyvalerolactone derivatives. Overall, several epicatechin and phenolic acid metabolites both of tissular and microbiota origin were the most representative markers of FB consumption. To our knowledge, this is one of the first studies where an untargeted LC-MS metabolomic approach has been applied in nutrition research on a grape-derived FB.