The Drug Development Process has the five steps: discovery and development, preclinical research, clinical research, regulatory body review and regulatory body post-marketing safety monitoring. According 3R principles: reduction, replacement, refinement, in silico methods are proposed for selected investigation data during preclinical and clinical phase of development of a new drug. The membrane permeability is one of the key properties when dealing with small molecules that have intracellular targets as their activity highly depends on their ability to cross the membrane. During the investigation of cytotoxicity of novel synthetic compound, it was found significant toxicity of ligand (S,S)-O,O-diethyl-ethylenediamine-N,N’-di-2-(3-cyclohexyl)propanoic acid dihydrochloride (DE-EDCP) toward glioma, melanoma, fibrosarcoma, and human leukemic cell lines. The in vitro studies, investigating membrane permeability of DE-EDCP, were performed using a parallel artificial membrane permeability assay (PAMPA). In addition to this, there is is an in-silico method, MembranePlus that can be used to assess and predict membrane permeability. In the present study we used the previously published in vitro data for new potential cytotoxicity active substance DE-EDCP, obtained by PAMPA method to validate in silico method, MembranePlus.