Trehalose is a disaccharide that is biosynthesized by many different organisms subjected to extreme conditions, such as dehydration, heat, oxidative stress, and freezing. This disaccharide allows organisms to better survive these environmental stresses; however, the mechanisms by which trehalose exerts its protective effects are not well understood. Methods to accurately measure trehalose from different organisms will help us gain better understanding of these protective mechanisms. In this study, three experimental approaches for the quantification of trehalose from biological samples were compared: an enzymatic trehalose assay (Trehalose Assay Kit; Megazyme International), a high-performance liquid chromatography coupled with refractive index detection-based assay, and a liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based assay. Limits of detection and quantification for each assay were compared, as were the dynamic ranges for all three assays. The percent recoveries for known amounts of trehalose spiked into bacterial and mammalian cellular lysates were also determined for each of the assays. Finally, endogenous trehalose produced by Escherichia coli cells was detected and quantified using these assays. Results from this study indicate that an LC–MS/MS-based assay is the most direct and sensitive method for the quantification of low concentrations of trehalose from biological samples; however, the enzymatic assay is suitable for the rapid quantification of higher concentrations of trehalose when an LC–MS/MS is unavailable.