Abstract The potential of drift tube ion mobility (IM) spectrometry in combination with high performance liquid chromatography (LC) and mass spectrometry (MS) for the metabonomic analysis of rat urine is reported. The combined LC–IM–MS approach using quadrupole/time-of-flight mass spectrometry with electrospray ionisation, uses gas-phase analyte characterisation based on both mass-to-charge ( m/ z) ratio and relative gas-phase mobility (drift time) following LC separation. The technique allowed the acquisition of nested data sets, with mass spectra acquired at regular intervals (65 μs) during each IMS separation (∼13 ms) and several IMS spectra acquired during the elution of a single LC peak, without increasing the overall analysis time compared to LC–MS. Preliminary results indicate that spectral quality is improved when using LC–IM–MS, compared to direct injection IM–MS, for which significant ion suppression effects were observed in the electrospray ion source. The use of reversed-phase LC employing fast gradient elution reduced sample preparation to a minimum, whilst maintaining the potential for high throughput analysis. Data mining allowed information on specific analytes to be extracted from the complex metabonomic data set. LC–IM–MS based approaches may have a useful role in metabonomic analyses by introducing an additional discriminatory dimension of ion mobility (drift time).