The chromatographic behavior and lipophilicity of 21 newly synthesized 3-(4- substituted benzyl)cycloalkanespiro-5-hydantoins were studied in reversedphase liquid chromatography conditions. Chromatographic behavior was analyzed by using thin-layer liquid chromatography and high-performance liquid chromatography. Chemically modified silica gel with covalently bonded C-18 groups was used as the stationary phase. Two-component mixtures of water and organic solvent in different volume ratios were used as mobile phases. In thin-layer chromatography, the mobile phases contained one of the following organic solvents: ethanol, n-propanol, i-propanol, t-butanol, acetonitrile, acetone, tetrahydrofuran, or 1,4-dioxane, while in highperformance liquid chromatography the mixture of water and acetonitrile was used as mobile phase. Based on the test results, the influence of the nature of the organic solvent in the mobile phase, the influence of the structure of spirohydantoin, as well as the influence of the content of the organic solvent on the chromatographic behavior of the tested compounds was analyzed. The linear dependence of the RM value on the volume fraction of organic solvent in the mobile phase was determined for all tested compounds. In this way, it was possible to calculate the chromatographic parameters of lipophilicity (RM0, ie log kw). The calculated chromatographic parameters of lipophilicity were compared with the calculated parameters of lipophilicity (log P) using linear regression analysis and linear dependences were established. The partition coefficients log P for all tested spirohydantoins were determined indirectly, as well, with the help of reference compounds, based on calibration models. LSER modeling was used to determine which interactions lead to the partition in the chromatographic systems, that is the partition between the mobile and stationary phases. In addition to identifying interactions, LSER modeling was used to determine the type as well as the strength of chemical interactions. QSRR analysis was applied to find mathematical models for describing the relationship between chromatographic lipophilicity parameters and the structure of newly synthesized spirohydantoin derivatives. The corresponding QSRR models were obtained by using the multiple linear regression (MLR) method. The predictability of each model is defined by an internal validation coefficient. Lipinski’s rule was applied to verify whether the tested spirohydantoins have good (theoretical) oral bioavailability. The influence of the organic solvent in the mobile phase, as well as the influence of the substituent on chromatographic lipophilicity, was assessed using multivariate analysis methods, hierarchical cluster analysis (HCA), and principal components analysis (PCA). These methods group the tested spirohydantoin derivatives, as well as the applied mobile phases based on their polarity. The relationship between chromatographic parameters of lipophilicity and the potential biological activity of spirohydantoin was analyzed using these two methods.