High mobility group box 1 (HMGB1) exhibits unique biochemical functions as a biologically intrinsic requisite factor and as a toxin. As such, it is imperative to understand the mechanism by which these seemingly and diametrically opposed functions are exerted. To effectively discriminate these actions is important to accurately and precisely determine the concentration of HMGB1 in biological samples. Research in this fascinating field, however, has been lacking due to the absence of a simple analytical system for HMGB1 that can be adapted for large sample numbers. In this report, we review the physiological and pathological significance of HMGB1 and describe the development of an assay method for this pleiotropic protein.