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Abstract In this study GC–MS-based untargeted metabolomics was used to identify the metabolic response of earthworm; Eudrilus eugeniae exposed to sub-lethal concentrations of chlorpyrifos-CHL, cypermethrin-CYP, Glyphosate-GLY, and Combined-C (all three pesticides) at the concentrations of 3, 6, and 12 mg/kg. Principal component analysis of the obtained datasets revealed a clear distinction between the control and treatment groups. The mean weight of the worms in the treated groups decreased significantly (p < 0.05). Among the identified metabolites, oleic acid (~ 93.47%), lysine (~ 92.20%), glutamic acid (~ 91.81%), leucine (~ 90.20%), asparagine (~ 94.20%), methionine (~ 92.27%), malic acid (~ 93.37%), turanose (~ 95.04%), maltose (~ 92.36%), cholesta-3,5-diene (~ 86.11%), galactose (~ 93.20%), cholesterol (~ 91.56%), tocopherol (~ 85.09%), decreased significantly (p < 0.05), whereas myoinositol (~ 83%) and isoleucine (78.09%) increased significantly (p < 0.05) upon exposure to the CHL, CYP, GLY, and C. Overall, the findings suggest that earthworms might be a new entry point for the pesticides into the food chain. The present study highlights that metabolomics can be a reliable approach to understand the effect of different xenobiotics including pesticides on the metabolic response of earthworms.