A simple and novel method for the direct determination of 2-deoxy D-glucose (2DG) is developed, using a hydrophilic interaction chromatography (HILIC) and evaporative light scattering detection (ELSD). Altech Altima HP HILIC column (250 x 4.6 mm, 5μ particles) was used along with the mobile phase consisted of water and acetonitrile in a linear inverse gradient elution. The proposed method is capable of separating 2DG from its precursor D-Glucal (DGL) and starting material D-Glucose (DGS) with resolution > 3.0. ELSD parameters such as nebuliser temperature and impactor position were found to be critical while detecting DGL. The optimized ELSD parameters were: impactor ON position, nebuliser temperature: 30°C, nitrogen flow rate 1.5 mL/min and gain1. Power regression curves were obtained in the range of 250-750μg/mL with R2 > 0.999. The percentage recovery of 2DG, DGL and DGS were found to be 99.5±0.7%, 101.1± 2.1, 98.5± 2.2 respectively at 95% confidence interval. The limit of detection (LOD) of DGL and DGS were found to be 20 ng and 15 ng respectively.