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Metabolomics is a valuable tool to gain mechanistic insight into biological processes. It is frequently used to obtain complementary details to other ‘omics technologies such as transcriptomics or proteomics. For knowledge generation, reproducible measurements of physiological, intact, and artifact-free metabolite levels are imperative necessitating further standardization of best practices to improve reliability of research outcomes. Here we report a novel cell sample preparation method (MxP® CellCollect) for metabolomics applications using adherent mammalian cells, which reduces the time consumption and physiological stress of conventional methods such as trypsinization or cell scraping. The most common sampling procedures to detach cells from their growth surface, trypsinization and scraping were compared to the MxP® CellCollect method investigating metabolite profiles of two breast cancer cell lines (MDA-MB-231 and MCF7). Metabolite levels as well as direction of metabolite changes differed tremendously revealing issues with trypsinization and scraping risking non-physiological or misleading results in contrast to MxP® CellCollect. Differences in metabolic profiles of cells harvested by trypsinization as compared to MxP® CellCollect or scraping can be directly attributed to prolonged, medium-free incubation time during cell detachment leading to a severely energy-depleted intracellular state. Labile metabolites or metabolites with fast intracellular turnover rates such as glycolysis and TCA cycle intermediates were strongly and significantly decreased by trypsinization. The same was true for amino acids and nucleoside triphosphates. Results obtained with scraping using methanol as solvent were multifaceted. Even mild evaporation of methanol prior metabolite extraction led to temperature- and/or light-dependent degradation of labile metabolites such as nucleoside triphosphates into di- and monophosphates liberating pyrophosphate. Furthermore, lipid metabolites, in particular cell membrane lipids, were found to have significantly lower levels than measured by trypsinization or MxP® CellCollect, indicating that lipid metabolites are insufficiently detached and/or unspecifically adsorb to the hydrophobic dish and scraping tool.