Obtained benzimidazole derivatives, our next synthesized heterocyclic compounds, belong to a new group of chemical bondings with potential anticancer properties (Błaszczak-Świątkiewicz & Mikiciuk-Olasik, 2006, J Liguid Chrom Rel Tech 29: 2367-2385; Błaszczak-Świątkiewicz & Mikiciuk-Olasik, 2008, Wiad Chem 62: 11-12, in Polish; Błaszczak-Świątkiewicz & Mikiciuk-Olasik, 2011, J Liguid Chrom Rel Tech 34: 1901-1912). We used HPLC analysis to determine stability of these compounds in 0.2% DMSO (dimethyl sulfoxide). Optimisation of the chromatographic system and validation of the established analytical method were performed. Reversed phases (RP-18) and a 1:1 mixture of acetate buffer (pH 4.5) and acetonitrile as a mobile phase were used for all the analysed compounds at a flow rate 1.0 mL/min. The eluted compounds were monitored using a UV detector, the wavelength was specific for compounds 6 and 9 and compounds 7 and 10. The retention time was specific for all four compounds. The used method was found to have linearity in the concentration range of (0.1 mg/mL-0.1 μg/mL) with a correlation coefficient not less than r(2)=0.9995. Statistical validation of the method proved it to be a simple, highly precise and accurate way to determine the stability of benzimidazole derivatives in 0.2% DMSO. The recoveries of all four compounds examined were in the range 99.24-100.00%. The developed HPLC analysis revealed that the compounds studied remain homogeneous in 0.2% DMSO for up to 96 h and that the analysed N-oxide benzimidazole derivatives do not disintegrate into their analogues - benzimidazole derivatives. Compounds 8, 6 and 9 exhibit the best cytotoxic properties under normoxic conditions when tested against cells of human malignant melanoma WM 115.