¹Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey DOI : 10.35333/jrp.2020.111 Metabolomics is one of the main areas to understand cellular process at molecular level by analyzing metabolites. In recent years, metabolomics has been emerged as key tool to understand molecular basis of disease, find diagnostic and prognostic biomarkers, and develop new treatment opportunities. One of the most important challenge for metabolomics analysis is sample complexity due to wide concentration dynamics. In untargeted metabolomics studies for human plasma samples, generally there is no pre-fractioning method for metabolites prior to LC/MS analysis. This situation causes problem for analysis of some specific metabolites belonging to different pathways in such a complex system. In this study, it was offered liquid-liquid extraction and ultrafiltration-based sample preparation technique for fractioning of non-polar metabolites. Human plasma metabolites were extracted with a well-known co-solvent system (methanol/water/chloroform) and separated according to their polarity in aqueous and organic phases. Ultrafiltration process was performed for both phases to clean-up the sample. Solvents were evaporated and the remaining parts were dissolved in an organic solvent, acetonitrile, to obtain the non-polar metabolite mixtures. Samples were analyzed using Q-TOF LC/MS system for metabolite profiling. Metabolites were separated in a C18 (Agilent Zorbax C18 1.8 μM, 50 x 2.1 mm) column at 0.200 ml min-1 flow rate. Water and acetonitrile mixture including 0.1% formic acid was used as the mobile phase in a 25 min gradient elution system. In the aqueous phase, 249 peaks were found and 60 peaks were obtained in the organic phase. 14 peaks were common in both phases. Results showed that co- solvent system could be used for pre-fractioning of metabolites and reduce complexity of human plasma for metabolite profiling. Keywords : Metabolomics; human plasma; XCMS; liquid-liquid extraction; ultrafiltration