AbstractBee pollinators are exposed to multiple natural and anthropogenic stressors. Understanding the effects of a single stressor in the complex environmental context of antagonistic/synergistic interactions is critical to pollinator monitoring and may serve as early warning system before a pollination crisis. This study aimed to methodically improve the diagnosis of bee stressors using a simultaneous untargeted and targeted metabolomics-based approach. Analysis of 84 Bombus terrestris hemolymph samples found 8 metabolites retained as potential biomarkers that showed excellent discrimination for nutritional stress. In parallel, 8 significantly altered metabolites, as revealed by targeted profiling, were also assigned as candidate biomarkers. Furthermore, machine learning algorithms were applied to the above-described two biomarker sets, whereby the untargeted eight components showed the best classification performance with sensitivity and specificity up to 99% and 100%, respectively. Based on pathway and biochemistry analysis, we propose that gluconeogenesis contributed significantly to blood sugar stability in bumblebees maintained on a low carbohydrate diet. Taken together, this study demonstrates that metabolomics-based biomarker discovery holds promising potential for improving bee health monitoring and to identify stressor related to energy intake and other environmental stressors.