Abstract: We present a method to directly observe three-dimensional (3D) distribution of aggregated gold nanoparticles (Au NPs) in live cells using dark-field sectional optical microscopy with chromatographic analysis. The scattering images of clustered Au NPs were transformed from RGB regular coordinate to hue, saturation, and value (HSV) cylindrical coordinate. The hue distinguished Au NPs from scattering images of cellular organelles. The saturation eliminated the interference from the defocused spots. The value, which indicated the scattering intensity, defined the 3D locations of Au NPs. The V/H ratio showed a linear relation with the Au NP number in the cluster. It was applied to estimate the numbers of aggregated nanoparticles in endosome vesicles. Using the chromatographic analysis, the calculated nanoparticle numbers were in good agreement with mass spectroscopic measurement. Compared to conventional methods, the chromatographic approach provides a simple observation of 3D distribution of Au NPs and their aggregation states in living cells.