Hexokinase (HK) is the first key enzyme in metabolic and glycolytic pathways. Aberrations in HK activity may result in various human diseases. In this study, we present a novel, one-step, simple, and rapid method for monitoring HK activity using a commercial personal glucose meter (PGM). In this design, HK catalyzes glucose phosphorylation, forming phosphorylated glucose, which is not detected by a PGM. Accordingly, the amount of unphosphorylated glucose remaining in solution following the HK-catalyzed reaction, which is inversely related to HK activity, can be determined using a PGM. Owing to the favorable features of the PGM, such as its simple operation, small size, low cost, wide accessibility, and portability, this proposed assay has great potential for the point-of-care testing of HK activity.