Oxidative stress (OS) is a physiological mechanism that is induced by pollutants and can lead to pathophysiological conditions. Pollutants may be activated by phase I metabolism enzymes, becoming more reactive, inducing additional OS. Paraquat-induced OS was measured in Caenorhabditis elegans that were modified to express CYP2E1—a human cytochrome P450 enzyme that bioactivates xenobiotics—in either their endoplasmic reticulum (erCYP2E1) or mitochondria (mtCYP2E1). Paraquat induced worm death, but deaths were reduced when worms were incubated in antioxidants. Reactive oxygen species significantly increased in mtCYP2E1 and wild-type worms, and significantly decreased in erCYP2E1 worms. These assays will validate a final OS testing method: monitoring global oxylipin production by tandem mass spectrometry. Oxylipins are potent signalling molecules in stress responses, and optimization efforts increased their detection by 66%. Further oxylipin testing will provide a mechanistic overview of the OS response which could explain differences in CYP2E1 isozyme activation.