In this application, 6 various mixed-mode phases were screened for separation of two neutral compounds. Experiments were conducted on four cation-exchange mixed-mode HPLC columns (Primesep P, Primesep 200, Primesep 100, and Primesep C) and two anion-exchange mixed-mode columns (Primesep D and Primesep B2). All phases showed different selectivity towards HPLC separation of prednisolone and glucose pentaacetate. Application shows that mixed-mode chromatography is a valuable tool in separation of neutral compounds by reversed-phase mechanism. Compounds were monitored by ELSD.