Metabonomics as an important component of functional genomics and system biology has gained increasing interest in drug development and biomarker discovery. Comprehensive metabonomics investigations are primarily a challenge for analytical chemistry. The main advantages of liquid chromatography–mass spectrometry (LC-MS) technique include high sensitivity, high resolution, and wide dynamic range. LC-MS–based technology platforms combined with multivariate statistical methods are used to explore the complex metabolite patterns of biofluids. In this chapter, the methodology and application of high-performance liquid chromatography (HPLC)-MS–based metabonomics in biomarker discovery are reviewed.