In this study, the chromatographic behavior of basic nitrogen in the structure of synthesized compounds containing some benzimidazolium salts (SA-61, SA-63, SA-97) was determined in hydro-organic mixtures containing polar aprotic acetonitrile and polar protic water. Protonation constant ((Figure presented.) values of studied compounds in 40, 45, 50, and 55 % (v/v) acetonitrile-water binary mixtures at 37 °C were obtained by using the reverse phase liquid chromatography method. The method is based on a model that relates the chromatographic behavior of the determined analytes to pH. The relationship between the calculated (Figure presented.) values and the macroscopic properties of acetonitrile was investigated. The agreement between the pKa (Figure presented.) values in the water medium determined by this approach was determined by the t-test. Using Abraham's solute parameters, the (Figure presented.) values at 25 °C were also estimated without performing an experimental study. By using the (Figure presented.) value calculated for each compound, the ionization percentages were calculated at different pH (1–12) values with the Henderson-Hasselbalch equation. The above-mentioned compounds were also tested on human lung and liver cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method for 48 h. One of the compounds (SA-97) demonstrated antiproliferative effect against both cell lines screened.