Aim: The delivered nanoparticle dose to cells in vitro may depend on nanoparticle sedimentation rate. Here, the conditions under which optical absorption spectroscopy can be used to quantify the deposited nanoparticle dose in vitro are investigated. Materials & methods: Nanoparticle cytotoxicity in both upright and inverted cell culture orientations is studied in the presence and absence of serum. Results: Dissolvable nanoparticles, such as ZnO, exhibit no difference in upright and inverted cultures due to dissolved Zn²⁺ ions that dominate cytotoxicity. Insoluble nanoparticles, however, exhibit different sedimentation rates and deposited doses that are linked to the observed cytotoxicity. Conclusion: The combined use of upright–inverted cell orientations and optical absorption spectroscopy can provide a simple experimental approach to interpret in vitro nano–biointeractions.