In the understanding of colony loss phenomena, a worldwide crisis of honeybee colonies which has serious consequences for both apiculture and bee-pollinationdependent farm production, analytical chemistry can play an important role. For instance, rapid and accurate analytical procedures are currently required to better assess the effects of neonicotinoid insecticides on honeybee health. Since their introduction in agriculture, neonicotinoid insecticides have been blamed for being highly toxic to honeybees, possibly at the nanogram per bee level or lower. As a consequence, most of the analytical methods recently optimized have focused on the analysis of ultratraces of neonicotinoids using liquid chromatography–mass spectrometry techniques to study the effects of sublethal doses. However, recent evidences on two novel routes—seedling guttations and seed coating particulate, both associated with corn crops —that may expose honeybees to huge amounts of neonicotinoids in the field, with instantly lethal effects, suggest that selected procedures need optimizing. In the present work, a simplified ultra-high-performance liquid chromatography–diode-array detection method for the determination of neonicotinoids in single bees has been optimized and validated. The method ensures good selectivity, good accuracy, and adequate detection limits, which make it suitable for the purpose, while maintaining its ability to evaluate exposure variability of individual bees. It has been successfully applied to the analysis of bees in free flight over an experimental sowing field, with the bees therefore being exposed to seed coating particulate released by the pneumatic drilling machine.