Despite the recent crystallographic determination of the crystal structure of Au-25(SCH2CH2Ph)(18) clusters, the question-whether all thiolate-capped, 25-atom gold clusters adopt the same structure, regardless of the types of thiols (e.g., long-chain alkylthiols, aromatic thiols, or other functionalized ones)-still remains unanswered. To crystallize long-chain or bulky ligand (e.g., glutathione)-capped Au-25(SR)(18) clusters has proven to be difficult due to the major amorphousness caused by such ligands; therefore, one needs to seek other strategies to probe the structural information of such gold clusters. Herein, we report a strategy to probe the Au-25 core structure and surface thiolate ligand distribution by means of NMR in combination with mass spectrometry. We use glutathione-capped Au-25(SG)(18) clusters as an example to demonstrate the utility of this strategy. One-dimensional (1D) and two-dimensional (2D) correlation NMR spectroscopic investigation of Au-25(G)(18) reveals fine spectral features that explicitly indicate two types of surface binding modes of thiolates, which is consistent with the ligand distribution in the Au-25(SCH2CH2Ph)(18) cluster. Laser desorption ionization (LDI) mass spectrometry analysis shows that Au-25(SG)(18) exhibits an identical ionization and core fragmentation pattern with phenylethylthiolate-capped Au-25 clusters. The charge state of the native Au-25(SG)(18) clusters was determined to be -1 by comparing their optical spectrum with those of [Au-25(SCH2CH2Ph)(18)](q) of different charge states (q = -1, 0). Taken together, our results led to the conclusion that glutathione-capped Au-25(SG)(18) clusters indeed adopt the same structure as that of Au-25(SCH2CH2Ph)(18). This conclusion is also valid for other types of thiolate-capped Au-25 clusters, including hexyl- and dodecylthiolates. Interestingly, the chiral optical responses (e.g., circular dichroism (CD) signals in the visible wavelength region) from the Au-25(SG)18 clusters seem to be imparted by the chiral glutathione ligands because no similar CD signals were observed in Au-25(SCH2CH2Ph)(18).