This report describes the strategies for gas-phase chiral and structural characterization of biomolecules using mass spectrometry (MS) and ion mobility-MS (IM-MS) techniques. Because both MS and IM-MS do not directly provide chiral selectivity, methodologies for adding a chiral selector are discussed in the context of (i) host–guest (H–G) associations, (ii) diastereomeric collision-induced dissociation (CID) methods, (iii) ion–molecule reactions, and (iv) the kinetic method. MS techniques for the analysis of proteins and protein complexes are briefly described. New advances in performing rapid 2D gas-phase separations on the basis of IM-MS are reviewed with a particular emphasis on the different forms of IM instrumentation and how they are used for chiral and/or structural biomolecular studies. This report is not intended to be a comprehensive review of the field, but rather to underscore the contemporary techniques that are commonly or increasingly being used to complement measurements performed by chiroptical methodologies. Chirality 21:E253–E264, 2009. © 2009 Wiley-Liss, Inc.