Gold nanoclusters (AuNCs) with well-defined structure and arrangement possess particular physical and functional properties. AuNCs that differ only by less than 1 nm in diameter corresponding to one atomic layer show different structural, optical, and physicochemical properties in a size-dependent mode, making their analytical characterization a challenge. Herein we describe an integrative approach to characterization of larger aqueous AuNC (Au102-pMBA44, Au144pMBA60 and higher) selected by gel electrophoresis (PAGE). We employ UV-vis, dynamic light scattering, and zeta-potential in combination with high-performance analytical techniques such as multiwavelength analytical ultracentrifugation and electrospray ionization mass spectrometry were used to separate aqueous AuNCs and to determine their specific hydrodynamic diameter, partial abundance, molecular weight, and mass/charge ratios when present in a complex mixture of AuNCs containing Au102 (1.6 nm), Au144 (2 nm), and Au288-328 (2.5 nm). Advanced analytical electron microscopy imaging (spherical aberration corrected BF/HAADF-STEM at low voltages dose) also revealed the structures of discrete arrangements of gold nanocluster populations.