With the recent publication of the FDA guidance on metabolites in safety testing (MIST), a reflection is provided that describes the impact of this guidance on the processes of drug metabolite identification and quantification at various stages of drug development. First, a retrospective analysis is described that was conducted on 12 human absorption, metabolism, and excretion (AME) trials with the application of these MIST criteria. This analysis showed that the number of metabolites requiring identification, (semi)-quantification, and coverage in the toxicology species would substantially increase. However, a significant proportion of these metabolites were direct or indirect conjugates, a class of metabolites that was specifically addressed in the guidance as being largely innocuous. The nonconjugated metabolites were all covered in at least one toxicology animal species, with no need for additional safety evaluation. Second, analytical considerations pertaining to the efficient identification of metabolites are discussed. Topics include software-assisted detection and structural identification of metabolites, the emerging hyphenation of ultraperformance liquid chromatography (UPLC) with radioactivity detection, and the various ways to estimate metabolite abundance in the absence of an authentic standard. Technical aspects around the analysis of metabolite profiles are also presented, focusing on precautions to be taken in order not to introduce artifacts. Finally, a tiered approach for metabolite quantification is proposed, starting with quantification of metabolites prior to the multiple ascending dose study (MAD) in humans in only specific cases (Tier A). The following step is the identification and quantification of metabolites expected to be of pharmacological or toxicological relevance (based on MIST and other complementary criteria) in selected samples from the MAD study and preclinical studies in order to assess metabolite exposure coverage (Tier B). Finally, a metabolite quantification strategy for the studies after the MAD phase (Tier C) is proposed.