Derivative ultraviolet spectrophotometly (DS), high performance liquid chromatography (HPLC), and ultra performance liquid chromatography (UPLC) were used to determine Isoconazole nitrate (ISO) and Diflucortolone valerate (DIF) simultaneously. In improved derivative spectrophotometric method, the spectral interference problem of unknown ingredients in this cream was solved by a new approach. Quantitative determinations were performed 289.2 nm (2nd derivative, n=5) for ISO and 262 nm (2nd derivative, n=9) for DIF. Linear calibration ranges and recoveries were 100-500 mu g.mL(-1), 5-25 mu g.mL(-1) and 99.67%, 102.03% for ISO and DIF, respectively. In HPLC method, linear calibration ranges and recoveries were 15-240 mu g.mL(-1), 6-60 mu g.mL(-1), 98.46% 99.41%, for ISO and DIF, respectively. UPLC method was developed and linear calibrations ranges and recovery values were 1-200 mu g.mL(-1) for both ISO and DIF, and 98.37% - 96.49% for ISO and DIF, respectively. UPLC method reduced the analysis time significantly on the other hand organic solvent consumption decreased about 5 times with using less amount of sample.