ObjectivesGlyphosate (N-phosphonomethyl glycine) and its commercial herbicide formulations have been shown to exert toxicity via various mechanisms. It has been asserted that glyphosate substitutes for glycine in polypeptide chains leading to protein misfolding and toxicity. However, as no direct evidence exists for glycine to glyphosate substitution in proteins, including in mammalian organisms, we tested this claim by conducting a proteomics analysis of MDA-MB-231 human breast cancer cells grown in the presence of 100 mg/L glyphosate for 6 days. Protein extracts from three treated and three untreated cell cultures were analysed as one TMT-6plex labelled sample, to highlight a specific pattern (+/+/+/−/−/−) of reporter intensities for peptides bearing true glyphosate treatment induced-post translational modifications as well as allowing an investigation of the total proteome.ResultsComparative statistical analysis of global proteome changes between glyphosate treated and non-treated samples did not show significant differences. Crucially, filtering of data to focus analysis on peptides potentially bearing glycine for glyphosate replacement revealed that the TMT reporter intensity pattern of all candidates showed conclusively that they are all false discoveries, with none displaying the expected TMT pattern for such a substitution. Thus, the assertion that glyphosate substitutes for glycine in protein polypeptide chains is incorrect.