Matrix-assisted laser desorption/ionization-ion mobility-mass spectrometry (MALDI-IM-MS) was used to analyze low mass gold-thiolate fragments generated from thiolate-protected gold nanoparticles (AuNPs). This is the first report of using gas-phase structural separations by IM-MS for the characterization of AuNPs, revealing significant structural variation between organic and gold-thiolate ionic species. Through the separation of background chemical noise, gold-thiolate ion species corresponding to fragments from the AuNP surface can be isolated. In the negative ion mode, many of these fragments correlate to capping structural motifs observed in the literature. In the positive ion mode, the fragment ions do not correlate to predicted structural motifs, but are nearly identical to the positive ions generated from the gold-thiolate AuNP precursor complexes. This suggests that energetic processes during laser desorption/ionization induce a structural rearrangement in the capping gold-thiolate structure of the AuNP, resulting in the generation of positively charged gold-thiolate complexes similar to the precursors of AuNP formation by reduction and negatively charged complexes more representative of the AuNP surface.