A rapid, sensitive extraction method was developed using the mixture Methanol – Dichloromethane - Water (MDW) (0.3:4:1v/v/v) and MeOH-H2O phase was assayed for sugar analysis. Photodiode-array detection (DAD) has been used to prove the extracted compound is UV inactive, Preparative High-performance liquid chromatography (prep HPLC) with Evaporative Light Scattering Detector (ELSD) coupled to electro spray ionization mass spectrometric (ESI-MS) detection in the positive ion mode gave MS and MSn fragmentation data which were employed for their structural characterization and separation of individual components. The various standard sugars were spotted using the solvent system n-butanol - acetone - diethylamine - water (10:10:2:6, v/v/v/v) in the cellulose layer for TLC analysis which indicated the presence of lactose, sucrose and glucose. This is the first assay of the sugar profile of the non-edible portion of musk melon, which can be further developed for characterization and evaluation of their quality with regards to their sugar composition.