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Simple Summary: Honeybees are vital pollinators, essential for maintaining ecosystems and biodiversity, but there is rising concern about the health of managed honey bee colonies, especially in heavily human-influenced ecosystems. Multiple factors contribute to this decline, including environmental conditions, forage quality, and pesticide use. These elements have a complex effect on the health and nutritional status of honeybee colonies and influence their response to disease and various stressors. In this study, the authors propose a new approach to assess colony health by correlating common measures of colony strength such as honey and pollen reserves, the number of bees, and brood with hemolymph proteins, common bee pathogens (Varroa destructor and Nosema spp.), and essential trace elements (iron, zinc, and copper). Significant correlations were found between hemolymph proteins and colony performance measures, and between V. destructor and hemolymph proteins and iron content. In conclusion, this study confirms the need for a more holistic approach to honeybee health considering all the relevant aspects and critical points that may affect colony survival. Environment, forage quality, management practices, pathogens, and pesticides influence honeybee responses to stressors. This study proposes an innovative approach to assess colony health and performance using molecular diagnostic tools by correlating hemolymph proteins with common measures of colony strength, prevalent honeybee pathogens (Varroa destructor and Nosema spp.), and essential trace elements (iron, zinc and copper). Colonies were selected from four apiaries located in different environmental and foraging conditions in the province of Bologna (Italy). Hemolymph samples were taken from June to October 2019. The Varroa infestation of the colonies was estimated by assessing the natural mortality of the mites, while the bees were tested for Nosema spp. spores using a microscopic method. Hemolymph proteins were quantified and separated using SDS-PAGE, and colony performance was assessed by determining adult bees, total brood, honey, and pollen reserves. The biomarkers measured proved to be useful for monitoring changes in performance and trophic conditions during summer and early autumn. Significant correlations were found between hemolymph proteins and colony performance measures. A positive correlation between pollen reserves, vitellogenin, and hexamerin 70a highlights the importance of these proteins for successful overwintering. In October, Varroa infestation was negatively correlated with total proteins, vitellogenin, apolipophorin II, transferrin, and hexamerin 70a, with negative implications for overwintering; furthermore, Varroa infestation was also negatively correlated with iron content, potentially affecting iron homeostasis. [ABSTRACT FROM AUTHOR]