Trehalose is a disaccharide biosynthesized by plants, fungi, and bacteria that acts as a natural cellular protectant when these organisms are subjected to harsh environmental conditions such as freezing or drying. In these organisms, trehalose is hypothesized to replace the water inside the cell, thus preventing proteins from unfolding and cell membranes from rupturing. Methods to detect and accurately measure trehalose from different organisms will help us gain a better understanding of the mechanisms behind trehalose’s ability to act as a cellular protectant. We have optimized a high-performance liquid chromatography (HPLC) method for the detection and quantification of trehalose from biological samples. This method uses a commercially available LC column for separation and a refractive index detector for the detection and quantification of trehalose. This assay was used to accurately detect and quantify trehalose levels from a variety of different sources. The HPLC assay reported here will be useful for the detection and quantification of trehalose from different biological sources, thus aiding in the study of the role that trehalose plays in cellular protection.