Retention parameters of psychotropic drug standards were determined on different columns, i.e., Octadecyl silica, Phenyl, Phenyl-Hexyl, Polar Reverse Phase, Pentafluorophenyl, and Cyanopropyl using aqueous eluent systems containing methanol or acetonitrile as organic modifiers, acetate buffer at pH 3.5 and addition of silanol blocker − diethylamine (DEA).The retention, separation selectivity, and sequence of elution were different when using eluents containing various organic modifiers. The significant differences were observed in retention parameters with a change of the used stationary phase. The various properties of stationary phases resulted in differences in analyte retention, peaks shape, systems efficiency and separation selectivity. The best shape of peaks were on Cyanopropyl (CN) column and the highest efficiency for most investigated psychotropic drugs were obtained on Phenyl-Hexyl and Polar RP columns.