New synthesized 1,4-disubstituted thiosemicarbazide derivatives were analyzed in the RP system, modified with the addition of salts; chaotropic (sodium hexafluorophosphate – Na PF6), cosmotropic (sodium phosphate – NaH2PO4), and neutral (NaCl) on Zorbax XDB C18 column. The effect of the eluent composition on the analytes retention (k), system efficiency (N), peak symmetry (As), and LOD values were all examined and compared to unmodified organic-aqueous mobile phase system. It was established that eluent modified with chaotropic salts addition was also the most advantageous according to other peak parameters such as the theoretical plates numbers and asymmetry factors. The lower LOD values were achieved in comparison to unmodified organic-aqueous eluent system. Compatibility of lipophilicity parameters calculated by the use of computer software with experimental ones measured by RP-HPLC was also the best for chaotropic modified mobile phase. To explain the observed phenomena, molecular modeling was performed for chosen representative compound in different environment representing examined mobile phase composition.