Metabonomics aims at the comprehensive and quantitative analysis of wide arrays of metabolites in biological samples. It has shown particular promise in the areas of toxicology and drug development, functional genomics, systems biology, and clinical diagnosis. Comprehensive metabonomics investigations are primarily a challenge for analytical chemistry. High-performance liquid chromatography-mass spectrometry (HPLC-MS) is an established technology in drug metabolite analysis and is now expanding into endogenous metabolite research. Its main advantages include wide dynamic range, reproducible quantitative analysis, and the ability to analyze biofluids with extreme molecular complexity. The aims of developing HPLC-MS for metabonomics range from understanding basic biochemistry to biomarker discovery and the structural characterization of physiologically important metabolites. In this review, the strategy and application of HPLC-MS-based metabonomics are reviewed.