The technique developed from the coupling of a separation technique and an on-line spectroscopic detection technology is known as hyphenated technique. The remarkable improvements in hyphenated analytical methods over the last two decades have significantly broadened their applications in the analysis of biomaterials, especially natural products. In this chapter, recent advances in the applications of various hyphenated techniques, e.g., GC-MS, LC-PDA, LC-MS, LC-FTIR, LC-NMR, LC-NMR-MS, and CE-MS, in the context of preisolation analyses of crude extracts or fraction from various natural sources, isolation and on-line detection of natural products, chemotaxonomic studies, chemical fingerprinting, quality control of herbal products, dereplication of natural products, and metabolomic studies are discussed with appropriate examples. Particular emphasis is given on the hyphenated techniques that involve liquid chromatography, as the separation tool.