Recent research has suggested that neonicotinoid insecticides applied to crops can be detected in adjacent pollinator-attractive habitats and pollen collected by honey bees. Honey bees, native bees, and monarchs could be exposed to neonicotinoids through ingestion of contaminated pollen, nectar, and milkweed leaves. Although these studies indicate that neonicotinoids can be detected in pollinator-attractive habitats, the magnitude and extent of potential adverse effects to honey bees, native bees and monarch larvae is an active area of research. In this study, we developed and validated an innovative method to simultaneously evaluate concentrations of clothianidin, imidacloprid, and thiamethoxam, and two imidacloprid metabolites (5-hydroxy imidacloprid and imidacloprid olefin) in plant foliage. The results indicate acceptable ranges for the recovery, low and high quality control (between 75% and 110%), while the matrix effect indicates minimal ion suppression or ion enhancement for all analytes. The calibration curves were linear over the concentration ranges with r2 at > 0.998. The data obtained established a validated, single extraction and LC-MS/MS analytical method for quantifying neonicotinoid concentrations to a method detection limit of 0.04 to 0.3 ng/g plant material. This method is comparable or in some cases better than existing methods that require separate extraction and/or LC-MS/MS methods. We are using the method to analyze leaf and pollen samples collected in 2017 from habitat patches adjacent to corn and soybean planted with neonicotinoid-treated seeds. This data will provide insights on spatio-temporal variability of neonicotinoid concentrations in Midwestern agroecosystems and help inform pollinator risk assessments.