Nowadays, metabonomics has been widely applied to the area of biomedicine. Based on metabolic profiling analysis, metabolomic studies can provide information of the metabolic phenotype affected by genetic or environmental factors. Liquid chromatography-mass spectrometry has been proven to be a robust platform for metabolic profiling by sensitive measurement of low molecular weight compounds. However, this sensitive platform requires standard protocols in the preanalytical stage to avoid unwanted results caused by improper operations. Therefore, in this chapter, we will present a systemic protocol for the collection and preparation of biofluids and extracts, such as blood, urine, tissues, and cell lines, including the collection and storage of samples in the clinic and the extraction procedures in the laboratory.