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Lanthanide (Ln) exposure poses a serious health risk to animals and humans. In this study, we investigated the effect of 10−9–10−3 M La, Ce, Eu, and Yb exposure onto the viability of rat renal NRK-52E cells in dependence on Ln concentration, exposure time, and composition of the cell culture medium. Especially, the influence of fetal bovine serum (FBS) and citrate onto Ln cytotoxicity, solubility, and speciation was investigated. For this, in vitro cell viability studies using the XTT assay and fluorescence microscopic investigations were combined with solubility and speciation studies using TRLFS and ICP-MS, respectively. The theoretical Ln speciation was predicted using thermodynamic modeling. All Ln exhibit a concentration- and time-dependent effect on NRK-52E cells. FBS is the key parameter influencing both Ln solubility and cytotoxicity. We demonstrate that FBS is able to bind Ln3+ ions, thus, promoting solubility and reducing cytotoxicity after Ln exposure for 24 and 48 h. In contrast, citrate addition to the cell culture medium has no significant effect on Ln solubility and speciation nor cytotoxicity after Ln exposure for 24 and 48 h. However, a striking increase of cell viability is observable after Ln exposure for 8 h. Out of the four Ln elements under investigation, Ce is the most effective. Results from TRLFS and solubility measurements correlate well to those from in vitro cell culture experiments. In contrast, results from thermodynamic modeling do not correlate to TRLFS results, hence, demonstrating that big gaps in the database render this method, currently, inapplicable for the prediction of Ln speciation in cell culture media. Finally, this study demonstrates the importance and the synergistic effects of combining chemical and spectroscopic methods with cell culture techniques and biological methods.