Abstract: HPLC-MS/MS was applied to the determination of clobetasol propionate and levofloxacin hydrochloride in cream disinfection products. The sample was extracted with acetonitrile for 20 min. The extract obtained was separated by using Thermo Hypersil Gold chromatographic column as stationary phase, and mixtures of 0.1% (φ) formic acid solution and acetonitrile (mixed in various ratios) as mobile phase in gradient elution. ESI+ and multi-reactions monitoring were adopted in MS/MS. Linear relationships between values of peak area and mass concentration of the 2 compounds were kept in the same range of 0.50-100 μg·L-1, with detection limits (3S/N) in the range of 0.06-0.009 μg·kg-1. Tests for recovery were made by standard addition method at the concentration levels of 0.50, 5.0, 50.0 μg·L-1, giving values of recovery in the range of 90.0%-95.6%, and values of RSD′s (n=5) found were in the range of 1.9%-4.0%.