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Comprehensive metabolome analysis using mass spectrometry (MS) often results in a complex mass spectrum and difficult data analysis resulting from the signals of numerous small molecules in the metabolome. In addition, MS alone has difficulty measuring isobars and chiral, conformational and structural isomers. When a matrix‐assisted laser desorption ionization (MALDI) source is added, the difficulty and complexity are further increased. Signal interference between analyte signals and matrix ion signals produced by MALDI in the low mass region (<1500 Da) cause detection and/or identification of metabolites difficult by MS alone. However, ion mobility spectrometry (IMS) coupled with MS (IM–MS) provides a rapid analytical tool for measuring subtle structural differences in chemicals. IMS separates gas‐phase ions based on their size‐to‐charge ratio. This study, for the first time, reports the application of MALDI to the measurement of small molecules in a biological matrix by ion mobility‐time of flight mass spectrometry (IM‐TOFMS) and demonstrates the advantage of ion‐signal dispersion in the second dimension. Qualitative comparisons between metabolic profiling of the Escherichia colimetabolome by MALDI‐TOFMS, MALDI‐IM‐TOFMS and electrospray ionization (ESI)‐IM‐TOFMS are reported. Results demonstrate that mobility separation prior to mass analysis increases peak‐capacity through added dimensionality in measurement. Mobility separation also allows detection of metabolites in the matrix‐ion dominated low‐mass range (m/z< 1500 Da) by separating matrix signals from non‐matrix signals in mobility space. Copyright © 2010 John Wiley & Sons, Ltd.