Determination of the number-based size distribution of silica particles using the centrifugal field-flow fractionation (CF3) method was investigated. Since the accurate determination of the number-based size distribution of materials is essential in the fields of nanotechnology and biotechnology, the establishment of a robust evaluation method is attractive. We explored optimization of the fractionation conditions for CF3 using silica particles. Using pure water media as the eluent, a band broadening effect was clearly found, and this effect became stronger with higher initial centrifugal field strengths. After addition of 0.05 wt% aqueous FL-70 as a dispersant in the eluent, size fractionation could be performed effectively at higher centrifugal field strengths, providing excellent size separation results. After optimization of the CF3 separation condition, we determined the number-based size distribution of silica particles using three methods: FE-SEM only, CF3 with multi-angle light scattering (CF3-MALS), and a combined CF3 with FE-SEM method (CF3-FE-SEM). To meaningfully compare the CF3-MALS results with the other two methods, we transformed the light scattering intensity to particle numbers using Mie theory. The determined number-based mean sizes of silica particles by the three methods agreed well; however, the evaluated standard deviation of the number-based size distribution of silica particles by the CF3-MALS method was slightly different. This was attributed to the unreliable sizing by MALS of smaller sized particles or low particle concentrations. The combined CF3-FE-SEM method provided near equal accuracy as the costly FE-SEM only and allowed for a significantly faster methodology because CF3 separation reduced the number of silica particles required for an accurate sizing down to just 50 particles per fraction.