The cadre of small molecule metabolites in a cell, aka the “metabolome,” is the final output of the genome and ultimate determinant of cell phenotype (DNA ? RNA ? protein ? metabolite ? phenotype). In addition to a genetic contribution, the metabolome is conditioned by diet, lifestyle and composition of the microbiome - ultimately determining health vs. disease status. Therefore, the profiling of small molecule levels using mass spectrometry-based metabolomic analyses can contribute significantly to our understanding of disease processes, enable confident molecular diagnoses, monitor the efficacy of therapies, and serve many other biomedical applications. Here, we discuss applications and considerations for the use of targeted and untargeted metabolite profiling using mass spectrometry. This topic is presented in the context of three independent research studies, described herein. First, we describe the use of targeted metabolomics to characterize the endocannabinoid system in a mouse model of alcohol “binge” drinking. We show that anandamide, one of the primary endogenous agonists of the cannabinoid receptor, along with several other endocannabinoids, increase in response to acute alcohol withdrawal. Further, we consider the biological significance of these observations. Second, we describe the use of mass spectrometry-based untargeted metabolite profiling and stable isotope tracing for defining metabolic perturbations that occur in the setting of sporadic amyotrophic lateral sclerosis (sALS), considering discoveries made with patient-derived skin fibroblasts. Using a multiomic approach, this study identifies and characterizes a distinct subgroup of sALS patients, possessing fibroblasts that are typified by enhanced transsulfuration pathway activity and glucose hypermetabolism. We speculate that this sALS patient subclass will prove to be selectively responsive to anti-oxidant therapies and therefore a recognition of this patient subclass may allow for the future establishment of personalized medicines. Finally, we discuss efforts to characterize solute carrier 25 (SLC25) family mitochondrial transporters using CRISPR knockout cell lines and a heterologous bacterial overexpression system-based protocol. These studies utilize mass spectrometry and stable isotope-labeled compounds in attempt to recognize potential biological substrates for these transporters.