Pharmacokinetics plays a key role in drug design. In the past many of the failures of drug candidates in development programs or even withdrawal medicines from market was attributed to their undesirable pharmacokinetic properties. Therefore, in the modern drug design and development the preclinical pharmacokinetic evaluation should be performed in the earliest stages of drug research. Lipophilicity is well known as a prime physico-chemical descriptor of drugs with at least partially relevance to all of the pharmacokinetic parameters. Moreover, it plays a vital role in the absorption of orally administrated drugs, leading to enhanced oral bioavailability. Today, a great number of techniques and methods are used for lipophilicity determination, still a reversed-phase high-performance liquid chromatography (RP-HPLC) has been recognized as the most useful tool for high throughput screening. Due to many advantages of chromatographic columns with immobilized artificial membrane (IAM-HPLC), in the last years the use of conventional C18 column for determination of lipophilicity is significantly decreased. The IAM columns is an innovative approach to chromatography in which the stationary phase surface emulates the lipid environment of the cell membrane; thus comparing to the C18 columns more precisely mimic interactions between a compound and biological membranes. The aim of this study was to evaluate the applicability of both above mentioned methods for determination of lipophilicity on the cytostatic drug gemcitabine. Furthermore, the obtained results were compared with the log P values predicted by Molinspiration and ChemAxon software. The obtained results indicated different chromatographic behavior of gemcitabine on C18 and IAM columns. Both experimental and theoretical data propound the high hydrophilicity of investigated compound.