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Insects, due to their small size, have limited energy storage space, but they also have high metabolic rate, so their hemolymph sugars are incredibly dynamic and play a number of important physiological functional roles in maintaining energetic homeostasis. In contrast to vertebrates, trehalose is generally the primary sugar found in insect hemolymph, which is followed by glucose and fructose. Many analytical chemistry methods exist to measure sugars, yet a direct comparison of methods that can measure all three simultaneously, and trehalose in particular, from low sample volumes, are sparse. Using the honey bee as a model, we directly compare the leading current methods of using High Performance Liquid Chromatography (HPLC) with an evaporative light-scattering detector and Gas Chromatography coupled with Mass Spectrometry (GC-MS) to determine which method would be better for measuring trehalose, glucose, and fructose in terms of reproducibility, accuracy, and sensitivity. Furthermore, we injected the enzyme inhibitors trehalozin (a trehalase inhibitor) and sorbose (a trehalase p-synthase inhibitor) to manipulate the trehalose levels in honey bee foragers as a proof of concept that this sugar can be altered independently of hemolymph glucose and fructose levels. Overall the HPLC method was less reproducible for measuring fructose and glucose, and it also had lower sensitivity for measuring trehalose. Consequently, significant differences in trehalose levels within the forager class were only detected with the GC-MS and not the HPLC method. Lastly, using the GC-MS method in the follow up study we found that trehalozin and sorbose causes a significant increase and decrease of trehalose levels respectively, in forager honey bees, independent of the glucose and fructose levels, ten minutes after injection. Taken together, these methods will provide useful tools for future studies exploring the many different physiological functional roles that trehalose can play in maintaining insect energetic homeostasis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)