We demonstrate a high-resolution in situ experimental method for performing simultaneous size classification and characterization of functional gold nanoparticle clusters (GNCs) based on asymmetric-flow field flow fractionation (AFFF). Field emission scanning electron microscopy, atomic force microscopy, multi-angle light scattering (MALS), and in situ ultraviolet-visible optical spectroscopy provide complementary data and imagery confirming the cluster state (e.g., dimer, trimer, tetramer), packing structure, and purity of fractionated populations. An orthogonal analysis of GNC size distributions is obtained using electrospray-differential mobility analysis (ES-DMA). We find a linear correlation between the normalized MALS intensity (measured during AFFF elution) and the corresponding number concentration (measured by ES-DMA), establishing the capacity for AFFF to quantify the absolute number concentration of GNCs. The results and corresponding methodology summarized here provide the proof of concept for general applications involving the formation, isolation, and in situ analysis of both functional and adventitious nanoparticle clusters of finite size.