A continuous-flow fluorometric procedure for the determination of 2-deoxy-D-glucose (2DG) is described. The method utilizes Technicon Autoanalyzer equipment and modules, and is based on the acid-catalyzed condensation of 3,5-diaminobenzoic acid with 2DG. The procedure permits analysis of 20 samples/h, is sensitive to concentrations of 2DG as low as 0.2 mg/100 ml, and requires sample volumes of only 0.25 ml. 2DG can be quantitatively measured in serum samples or tissue extracts without requiring deproteinization. Glucose does not interfere with the assay while 2-deoxy-D-ribose develops a fluorescence which is about 15% of that produced by the same amount of 2DG and is additive when both deoxy sugars are present together. The procedure is accurate, reproducible, and fast, and can be run continuously.