Lignocellulosic biomass, found in wooden plant husks is a potent renewable material source which can be utilised to form various chemicals and biomaterials including polyols such as xylitol. Xylitol has been used commercially as an alternative to sucrose in many products as bulk sweetener in non-cariogenic confectionery as well as in diabetic diets and solutions for parenteral nutrition. Therefore, this study aims to optimise separation parameters of the Shimadzu high performance liquid chromatography (HPLC) (Model No: LC-20A) and quantify the potential of coconut husk as substrate for Candida albicans in producing xylitol using HPLC. Pretreatment to depolymerise components of biomass i.e. cellulose, hemicellulose and lignin were done using dilute acid hydrolysis method which yielded fermentable reducing sugars, xylose. Xylose is a monosaccharide with an aldehyde functional group, a reducing sugar which is then utilised and fermented by the yeast Candida albicans to form xylitol. In this study, the media used for fermenting pretreated coconut husk and Candida albicans is a synthetic defined (SD) minimal broth. Growth curves against concentration of reducing sugar were plotted to determine utilisation and production trends with specific mixes of carbon sources. The presence of reducing sugars were tested using 3,5-dinitrosalicylic acid (DNS) assay in pretreated coconut husk yielded 13.22 g/L. In order to analyse the product with HPLC, an existing protocol is modified for Hypersil GOLD™ C18 column with acetonitrile as the mobile phase. Results obtained from HPLC analyses using developed protocol suggested the formation of xylitol from the fermentation of pretreated husk by Candida albicans. These can be followed by purification of fermented media in obtaining a better separation of contaminating peaks.