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 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.