Metabolomics, or alternately metabonomics, an emerging field of biochemical research, is a complementary technique to genomics, transcriptomics, and proteomics. Direct quantitative measurements of metabolite expressions in urine, serum, plasma, and tissue are essential for the study of biological processes in normal and disease states. Since the number of metabolites in a biological sample is large separation science plays an important role in metabolomic research. This review describes the role of GC, HPLC, CE, and supercritical fluid chromatography in metabolomic research. The application of each technique is discussed and its advantages and limitations are discussed with selective illustrative examples.