Selected steroid compounds (androsterone, epi-androsterone, dehydro-epi-androsterone, testosterone, stigmasterol, β-sitosterol, estradiol, hydrocortisone and cholesterol) were separated by adsorption TLC on silica gel 60F₂₅₄ non-impregnated and impregnated with a 1% and 5% aqueous solution of CuSO₄. The mixture of chloroform and acetone in different volume compositions was used as mobile phase. On the basis of the research done herein, it was shown that it was not possible to separate all of the steroid compounds investigated herein by using the same chromatographic conditions. Best chromatographic conditions for the separations of particular pairs of substances were investigated. Impregnation of silica gel 60F₂₅₄ with 1% and 5% aqueous solutions of CuSO₄ influences the separation of particular pairs of studied steroid compounds. The obtained results indicate that cluster analysis can be an alternative method for the estimation of chromatographic separation of the studied steroid compounds. The resultant spectrodensitograms of the studied compounds indicate that applied sorbents (silica gel 60F₂₅₄ not impregnated and impregnated with CuSO₄) as well as the use of sulfuric acid as visualizing reagents have an influence on the wavelength of the obtained fundamental absorption band (λ_max) and the additional absorption bands, as well as on their intensity values [AU]. This fact indicates the need for standaridization of the spectrodensitometric investigations regarding the applied chromatographic conditions.