Chromatographic properties of seven steroids: estrogens (β-estradiol and estrone), androgens (testosterone, methyltestosterone, trans-androsterone), progesterone and cholesterol have been studied by planar chromatography with usage of High Performance Thin Layer Chromatography (HPTLC) and Thin Layer Chromatography (TLC) plates. Normal, reversed and cyano-bonded silica stationary phases were tested with five binary mobile phases (acetonitrile-water, acetonitrile-DMSO, acetonitrile-methanol, acetone-petroleum ether, acetone-water) in which the concentration of organic modifier varied from 0 to 100% (v/v). This study reports the optimization of steroid hormones separation. Principal Component Analysis (PCA) based on calculated molecular descriptors quantitatively differentiating solutes was performed in order to investigate the similarity and dissimilarity between tested compounds. The separation abilities of mobile and stationary phases were compared based on separation factor α. Chromatographic retention data and possible retention mechanisms also were discussed.