Introduction: Neonicotinoids, neuroactive insecticides that have replaced more hazardous carbamates and organophosphates, are widely used in agriculture, landscaping, and on household pets. Neonicotinoid insecticides including imidacloprid, acetamiprid, thiamethoxam, clothianidin and dinotefuran, which represent approximately 20% of the insecticides currently used in the USA, were detected in common food staples in the U.S. diet. Because of their systemic properties they cannot be washed off from fruits and vegetables. Results from previous studies estimate that approximately 49% of the U.S. general population is exposed to neonicotinoids. The fact that neonicotinoid metabolites were detected at higher concentrations and at wider concentration ranges than the corresponding parent compounds suggests metabolites are better biomarkers of exposure. Because neonicotinoid use is on the rise and their risk to humans is not well known, identifying suitable neonicotinoid metabolites that can potentially be used as biomarkers of exposure is critical to improve the exposure assessment process. Methods: We used in-vitro approaches to generate metabolites of imidacloprid, acetamiprid, thiamethoxam, clothianidin and dinotefuran. We used mass spectrometry to identify these novel neonicotinoid biomarkers. Results: We identified several in-vitro metabolites of imidacloprid, acetamiprid, thiamethoxam, clothianidin and dinotefuran. Most of these metabolites were the result of common detoxification reactions of human xenobiotics (e.g., demethylation, hydroxylation, guanidine- and urea-derivatives). Conclusions: The newly identified neonicotinoid metabolites will be used as biomarkers of exposure to neonicotinoids and incorporated in population-based studies such as the National Health and Nutrition Examination Survey to improve our understanding of the potential health hazards from exposure to this widely used class of insecticides. Disclaimer: The findings and conclusions in this abstract are those of the authors and do not necessarily represent the views of the CDC.