In this study, the HPLC, UV-vis, LC-MS, and 1H NMR characteristics of 14 different phase II mono- and mixed conjugates of quercetin were determined, providing a useful tool in the identification of quercetin phase II metabolite patterns in various biological systems. Using these data, the phase II metabolism of quercetin by different rat and human liver and intestine in vitro models, including cell lines, S9 samples, and hepatocytes, was investigated. A comparison of quercetin phase II metabolism between rat and human liver and intestinal cell lines, S9, and hepatocytes showed considerable variation in the nature and ratios of quercetin conjugate formation. It could be established that the intestine contributes significantly to the phase II metabolism of quercetin, especially to its sulfation, that organ-dependent phase II metabolism in rat and man differ significantly, and that human interindividual variation is higher for quercetin sulfation than for glucuronidation or methylation. Furthermore, quercetin conjugation by different in vitro models from corresponding origins may differ significantly. The identification of the various mono- and mixed quercetin phase II conjugates revealed significant differences in phase II conjugation by a variety of in vitro models and led to the conclusion that none of the in vitro models converted quercetin to a phase II metabolite mixture similar to the in vivo plasma metabolite pattern of quercetin. Altogether, the identification of a wide range of phase II metabolites of quercetin as presented in this study allows the determination of quercetin phase II biotransformation patterns and opens the way for a better-funded assessment of the biological activity of quercetin in a variety of biological systems.