The freely dissolved concentration in the assay medium (Cfree) and the total cellular concentration (Ccell) are essential input parameters for quantitative in vitro-to-in vivo extrapolations (QIVIVE), but available prediction tools for Cfree and Ccell have not been sufficiently validated with experimental data. In this study, medium-water distribution ratios (DFBS/w) and cell-water distribution ratios (Dcell/w) for four different cells lines were determined experimentally for 12 neutral and five ionizable chemicals. Literature data for seven organic acids were added to the dataset, leading to 24 chemicals in total. A mass balance model based on bovine serum albumin-water (DBSA/w) and liposome-water distribution ratios (Dlip/w) of the chemicals was used to calculate DFBS/w and Dcell/w. For all neutral and basic test chemicals, the mass balance model predicted DFBS/w and Dcell/w within a factor of 3 and 3.4, respectively, indicating that existing models can reliably predict Cfree and Ccell for these chemicals. For organic acids, a further refinement of the model will be required as large deviations between modeled and measured binding to assay medium and cells of up to a factor of 370 were found. Furthermore, saturation of medium proteins should be further explored for organic acids and neutral chemicals with moderate hydrophobicity.