The uridine diphosphate glucuronosyltransferases (UGTs) represent a super-family of Phase II membrane-bound drug metabolizing enzymes (MW = 50–60 kDa) that are responsible for the transfer of glucuronic acid from the cofactor uridine diphosphoglucuronic acid (UDPGA), to a nucleophilic acceptor substrate (Dutton 1980, 1997, Mackenzie et al. 1997, King et al. 2000, Tephly and Green 2000, Tukey and Strassburg 2000, Rowland et al. 2013). The resulting glucuronide conjugates are more water soluble than the parent drug molecule (aglycone), thus rendering them subject to facile excretion. UGT-catalyzed glucuronidation is generally accepted to be the second most prevalent metabolic pathway, with only cytochrome P450 oxidation contributing to a greater percentage of drugs cleared by metabolism (Williams et al. 2004). To date, more than 20 individual UGT isoforms have been identified in humans, with the majority of xenobiotic metabolism being catalyzed by isoforms in the UGT1A and UGT2B subfamilies. UGTs are also capable of catalyzing the addition of other sugar moieties such as glucose or xylose to an aglycone acceptor molecule (Mackenzie et al. 2008, 2011, Meech et al. 2015). Well-documented structural moieties that can serve as nucleophilic acceptors for glucuronidation, include alcohols (aromatic or aliphatic), carboxylic acids (resulting in the formation of acyl glucuronides), thiols, amines, and nucleophilic carbon atoms (Figure 5.1) (Richter et al. 1975, Sorich et al. 2006). This chapter will cover basic UGT enzymology, as well as the important role that UGTs play in drug metabolism, drug interactions and drug efficacy, and safety. UGT ontogeny, in vitro-in vivo extrapolation (IVIVE) for UGT substrates, analytical challenges with glucuronide metabolites and general experimental considerations with the UGTs will also be reviewed.