In this work, we present a new method for synthesis of silica gel stationary phases based on calixarene derivatives. In order to achieve it, 25,27-dipropoxy-26,28-bis-[3-propyloxydimethylsililoxy]calixarene has been synthesized in six steps and immobilized on silica via chlorotrimethylsilane. Stationary phases were characterized by elemental analysis, infrared spectroscopy and thermal analysis and used for the separation of amino acid derivatives by high performance liquid chromatography. The effect of isocratic and gradient elution, pH and column temperature on retention and selectivity of the Fmoc-protected amino acids were studied. The retention mechanism was also discussed. The results indicated that the stationary phase behaves like a reverse phase packing. Size exclusion, electron-π, π-π and hydrophobic interactions seem to be involved in the separation process.