In this study a novel, simple and rapid reversed-phase high performance liquid chromatography (HPLC) procedure for simultaneous determination of vitamins A and E (retinol and alpha-tocopherol) in blood serum has been developed and validated using monolithic column and diode-array detection (DAD).The monolithic column Chromolith Performance RP-18e (100mm×4.6mm) was operated at ambient temperature. One hundred percent methanol at flow rate 2.5mlmin −1 was used as a mobile phase. Detection of both compounds was performed with diode-array detector, retinol was monitored at 325nm and alpha-tocopherol at 295nm.The linear dependence between peak area and concentration ranged from 0.25 to 10.00μmoll −1 for retinol and 0.5–50.0μmoll −1 for alpha-tocopherol. The limit of detection (LOD) for retinol was 0.02μmoll −1 and limit of quantification (LOQ) was 0.07μmoll −1 . The limit of detection (LOD) for alpha-tocopherol was 0.1μmoll −1 and limit of quantification (LOQ) was 0.3μmoll −1 . Retinol was eluted in 0.8min and alpha-tocopherol in 1.4min. The simultaneous analysis of vitamin A and E can be achieved in less than 2min.The implementation of monolithic column Chromolith Performance shortens the time of analysis of both vitamins four times in comparison with using traditional particulate column Pecosphere C18 (150mm×4.6mm), 5μm. This fact may play an important role for routine clinical analysis of biological samples.