Many environmental toxicants can activate estrogen receptor α (ERα), disrupting normal endocrine function. While these activities are predicted across in silico, in vitro, and in vivo models, translating active concentrations between these systems remains challenging. We hypothesized that cellular uptake and the resulting free intracellular toxicant concentration could bridge this gap. Using cell-free (hER) and cell-based (ERα-CALUX cells) estrogen assays, we tested this hypothesis by determination of the free intracellular concentration available for binding to the intracellularly located ERα. Predictive modeling identified three classes of estrogenic chemicals from the ToxCast collection: bisphenols, parabens, and phthalates. Experimental data confirmed potency differences of up to 100-fold between the cell-free and cell-based models. Cellular toxicokinetic (TK) parameters, including cellular uptake and intracellular binding, were determined using computational and experimental methods. Incorporating experimental TK parameters significantly improved the correlation between ERα activities in the cell-free and cellular models (from r = 0.6230, P = 0.0989 without corrections to r = 0.8869, P = 0.0033 after corrections), and bridged the gap between the cell free and cell based assays. Both computational and experimental TK parameters varied widely across chemical classes and compounds. Correcting active concentrations for free intracellular levels enhanced assay correlations, with experimentally derived corrections showing the strongest improvement with r = 0.8869 (compared to the in silico derived corrections with r = 0.811). Our findings highlight the critical role of free intracellular concentration in determining the biological activity of estrogenic toxicants and emphasize its importance in accurately assessing their endocrine-disrupting potential.