The effects of temperature, organic modifier and the type of acid on the retention factor, the resolution and peak shape of betamethasone and dexamethasone are described. The study is performed using narrow bore porous graphitic carbon (PGC) columns online with diode-array detector (DAD) and ESI MS/MS. The results show that temperature affects the retention behaviour of the two compounds and ACN yields the best separation while no effect is obtained by changing the type of organic acid. The developed method is applied for the confirmation of dexamethasone and betamethasone in human urine.