In a complex and expensive process such as the development of a new drug, any improvement, simplification or acceleration of the process is worth a lot. Most of the candidate molecules are rejected already in the early stages of development due to unfavorable pharmacokinetic characteristics that are closely related to the physicochemical properties of the compound – lipophilicity, water solubility, binding affinity for proteins, affinity for biomembranes, etc. Therefore, methods used to determine such properties are of special importance. Among them are QSAR and QSPR computer methods that use computer algorithms to predict various properties of a molecule, as well as biomimetic chromatography methods that test such properties in vitro. This paper includes the pharmacokinetic characterization of two biologically active substances used in therapy of inflammatory bowel diseases - aloin and aloe emodin. They are characterized first by a series of QSPR methods that calculate their lipophilicity, solubility, human intestinal absorption, oral bioavailability and the percentage of binding to plasma proteins, and then by reversed-phase thin-layer chromatography and reversed-phase high-performance liquid chromatography that show the hydrophobicity of the analyte, high-performance liquid chromatography with a stationary phase containing immobilized artificial membranes showing lipophilicity, then with a stationary phase containing immobilized human serum albumin and finally one containing immobilized α1-acid glycoprotein to obtain the analyte binding affinity for HSA and AGP. In the end, it was shown that aloe emodin is more hydrophobic than aloin, is less soluble in water, has greater absorption in the intestine as well as oral bioavailability, and binds with greater affinity to plasma proteins compared to aloin. At the same time, deviations between different QSPR calculations are very big, and the most reliable pharmacokinetic properties remain to be investigated by in vitro methods.