Metabonomics focuses on metabolite profile changes in diverse living systems caused by a biological perturbation. These metabolite signatures can be achieved with techniques such as gas chromatography, high-performance liquid chromatography (ultra-high-performance/pressure liquid chromatography and capHPLC), capillary electrophoresis, and capillary electrochromatography normally hyphenated with MS. In this review we present the latest developments of the abovementioned techniques applied in the field of metabonomics, with applications covering phytochemistry, toxicology and clinical research.