Our study demonstrates that biomarker research using liquid chromatography (LC)-high resolution (HR) mass spectrometry (MS) based untargeted metabolomics profiling is susceptible to the discovery of false positive biomarkers. LC-MS, especially LC-HRMS, is popularly used to discover putative biomarkers through comparing untargeted metabolomic profiles between a patient group and a control group. This approach is susceptible to various pre-analytical, analytical, and post-analytical biases. Moreover, isotopes, adducts, in-source fragment products of some metabolites, artifacts, and contaminants could be wrongly considered as unique metabolomic features. To what extent the putative metabolomic biomarkers could be false remains unknown. We attempted to identify putative biomarkers for differentiating two artificial groups of plasma samples (12 samples in each group) with well-defined differences in their metabolome contents.