The in vitro cytotoxicity of nanomaterials is commonly assessed by monitoring cell viability under exposure to their suspensions. The actual nanoparticle dose delivered to cells for the same nominal nanoparticle concentration may depend on their settling rate. The goal here is to evaluate quantitatively the importance of settling for the in vitro cytotoxicity evaluation of aerosol-made, fractal-like SiO2 nanoparticles that have similar agglomerate size and structure but varying primary particle diameter. Their deposition rate is measured by UV-visible spectroscopy and their in vitro cytotoxicity is evaluated in both standard upright and inverted murine macrophage culture configurations. Silicas of larger primary particles settle faster and appear more cytotoxic than those of smaller ones in standard upright cell cultures. In contrast, with inverted cell cultures all silicas show similar and much lower cytotoxicity revealing the impact of nanoparticle settling in such assessments that are benchmarked with H2O2 solutions and nanosilver suspensions. When the measured settled fractions are accounted for, the reduction in cell viability depends on the surface of SiO2 nanoparticles deposited onto cells regardless of primary particle size. This study quantitatively demonstrates the importance of nanoparticle settling for in vitro studies of the widely used fumed SiO2 and highlights that settling measurements combined with inverted-upright cell cultures can be useful tools in nanotoxicology studies.