While the metabolism of small molecule drugs has been dominated by the cytochrome P450 family of enzymes, many other enzyme families exist that help facilitate the conversion of lipophilic drug molecules to metabolites that may be readily excreted from the body. A shift in the chemical space that medicinal chemists are interrogating has led to generally more polar drug molecules, which has in turn has caused an increase in the prevalence of non-cytochrome P450 metabolic pathways. It is thus critical that drug metabolism scientists are aware of in vitro methods for identifying the role of these enzymes. For example, the role of the thermally labile metabolic enzyme flavin monooxygenase (FMO) is likely under-diagnosed due to the way in which in vitro incubations in human liver microsomes are conducted, with pre-incubations at 37 °C often devoid of NADPH. In addition, interest in the oxidative enzyme aldehyde oxidase (AO) has surged in recent years in response to its direct negative impact on clinical programs. Lastly, the UDP-glucuronosyltransferase (UGT) family of enzymes are highly problematic, with the extrapolation from in vitro systems to predict clearance to in vivo being a challenge. While many non-cytochrome P450 enzymes exist, the focus of this chapter will be on these three important enzyme systems.