To advance the information content in biotechnology and nanotechnology studies using mass spectrometry (MS) techniques, two-dimensional (2D) post-ionization separations combining ion mobility (IM) with time-of-flight MS (IM-TOFMS) are described. The added data dimensionality consists of ion structural information based on the ion collision cross-section determined by IM with accurate mass measurements obtained by TOFMS. The basic theory of IM and practical implementation in IM-MS instrumentation is discussed and described in the context of high throughput separations (µs-ms), peptide and post-translational modification (PTM) identification, and structural determinations, for applications ranging from proteomics and tissue imaging, to characterization of carbon nanotubes.