Glucose is a primary source of energy used in most organisms. Thus, development of reliable approaches to measure intracellular glucose uptake is an important research issue. 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG), as a fluorescent glucose derivative, has been widely used to track intracellular glucose uptake by fluorescence imaging and measuring in mammalian cells. However, the avoid-less cross-interference of intrinsic autofluorescence background and tested fluorescent compounds limits its ability to provide trustworthy information on intracellular glucose uptake. By the extraction, separation and detection of 2-NBDG, a simple, sensitive and accurate HPLC-FLD method was established and validated for the measurement of intracellular glucose uptake in HepG2 cells. The developed method has been employed successfully to assess the glucose uptake activity of anti-diabetic drugs and fluorescent natural products. A fit-for-purpose partial validation was further performed for quantification and comparison of glucose uptake in AML12, LO2 hepatocytes, L6 myoblasts and 3T3-L1 preadipocytes.