Metabolomics is a comprehensive profiling tool used to identify qualitatively and quantitatively all the metabolites present in a biological system. As the number of metabolites in a living being is assumed to be around 30,000, it is necessary to use an adequate extraction procedure and a good analytical technique to perform metabolomic analyses. Each analytical platform used in metabolomics has both advantages and disadvantages in terms of the sensitivity and resolution for metabolites to be detected. Of the methods, nuclear magnetic resonance (NMR) spectroscopy has been proved to have several advantages over other MS-based methods. NMR provides an efficient, robust, and nondestructive metabolomics analysis of crude extracts or samples as well as easy quantitation without calibration curves for each metabolite, although it shows relatively low sensitivity than MS-based methods. Thus, NMR-based metabolomics have been often used for the first step to capture the insight of the metabolome of organisms. This chapter presents general steps of NMR-based metabolomic analysis involved in the study of biodiversity.