Identifying and quantifying a wide variety of metabolites requires appropriate analytical technologies such as NMR, gas chromatography, and/or LC-MS. . LC-MS has become the most widely used technology for untargeted metabolomics. However, with the exception of human biofluids, standardized metabolomic sample preparation practices and protocols are not well established and vary greatly between labs. Standardized protocols are inherent to the collection and interpretation of high-quality data, the reproducibility of experiments, and the capability to compare results across studies or laboratories. There have been numerous publications and reviews demonstrating and collating sample preparation optimization methods for human biofluids and other matrices, however, as the field, technologies, and hypotheses evolve there is a need to adopt systematic, scalable, and high-throughput experimental design approaches to re-examine, develop, and standardized methodologies. Metabolome coverage, selectivity, reproducibility, and the introduction of artifacts depend upon the preanalytical protocols employed. Different extraction methods result in variable metabolite recovery due to the complex chemical space of metabolism. Conditions such as temperature, time, pH, and solvent composition (among others) directly affect the metabolites that can be detected and interpreted. Thus, the extraction protocol plays a critical role in determining the scope of metabolites that can be studied and affects downstream steps such as statistical analysis and the overall biological conclusions drawn from an experiment.