Ellagitannins and ellagic acid (EA) are metabolized by the gut microbiota to produce urolithins that could be responsible for the health effects attributed to ellagitannin-containing food products. Several urolithin aglycones could be present in fecal samples while glucuronide and sulphate conjugates are mainly found in plasma and urine. So far, the lack of available standards has made difficult their correct identification and quantification. In the present study, UV and MS spectra characteristics of urolithins and their phase II metabolites have been determined using different systems based on liquid chromatography (LC) coupled with diode-array or mass spectrometer detectors with different analyzers (triple quadrupole (QqQ) and quadrupole time-of-flight (QTOF)). Chromatographic separation was achieved on a reversed-phase Poroshell C18 column (3×100mm, 2.7μm). Elution order, characteristic UV spectra, and relative response factors (RRFs) with respect to their parental compound (EA) and the most common metabolite urolithin A (Uro-A) were determined. This contribution, along with the most important mass spectra characteristics (MRM transitions, qualifier/quantifier ratio, accurate mass and fragmentation pattern) will allow the determination of urolithin metabolites in different biological samples and their quantification even if not all metabolites are commercially available. The methods developed in the three systems have been fully validated in terms of linearity, sensitivity, precision, recovery, matrix effect, selectivity and stability. After that, they were successfully applied to complex biological matrices (urine, feces and plasma) from two human studies in which volunteers consumed ellagitannin-containing foods, such as walnuts and pomegranate extracts.