The development and use of HPLC-MS for the study of metabonomics is reviewed. To date the technique has been applied to the analysis of urine samples obtained from studies in rodents in investigations of physiological variation (e.g., factors such as strain, gender, diurnal variation, etc.) and toxicity. Examples are provided of the use of conventional HPLC, capillary methods and the recently introduced high-resolution systems based on a combination of high pressure and small particle size ("UPLC"). Comparison is also made of the use of 1H NMR spectroscopy and HPLC-MS for the analysis of biofluid samples and the advantages and limitations of the two approaches are assessed. Likely future developments are considered.