Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study, we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single, living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique, it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research.