Metabonomics is a subject of comparative analysis of metabolites in a series of similar biological samples. Metabolites play a vital role in biological systems, so metabonomics can be used to find and identify biomarkers, or to better understand the effects of drugs or diseases on known and unknown biological pathways. Successful metabonomics research depends on effective metabolite extraction. For non-targeted metabonomics research, it is necessary to extract a variety of metabolites from cells and body fluids, and remove protein and other components that do not need to be analyzed. In addition, the physical and chemical properties of metabolites are diverse, and the abundance varies by several orders of magnitude, which further increases the difficulty of extraction. Liquid-liquid extraction is a two-phase separation method, which is often used to extract metabolites. The properties, volume, solvent ratio and pH value of organic solvents and aqueous solutions in liquid-liquid extraction must be carefully considered. These factors will significantly affect the amount of metabolites extracted and the repeatability of the experiment. This application report introduces the method of extracting red blood cell metabolites by liquid-liquid extraction. The results show that it is very important to adjust the ratio of water phase to organic phase for two-phase separation. At the same time, the pH value of water phase also has a great influence on the extraction amount of metabolites. In order to extract as many metabolites as possible, it is necessary to use multiple pH values for extraction.