Absorbance ratio is a method that has been developed in the simultaneous determination of UV spectrophotometry. There are no research reports on this method on simultaneous estimation of betamethasone valerate and neomycin sulfate mixture in cream preparations. Therefore, this study aims to develop and validate the absorbance ratio method for the simultaneous estimation of betamethasone valerate and neomycin sulfate mixture in cream preparations with 70% ethanol as a solvent and at the intersection of wavelength 245 nm. The result showed that the regression equation for BTS is Y = 0.04039 X + 0.00349 and the regression equation for neomycin sulfate is Y = 0.00254 X + 0.00165 with validation tests were LOD was 0.645 µg/mL, LOQ was 2.15 µg/mL, accuracy was 100.07%, precision was 0.87% for betamethasone valerate and LOD was 2.215 µg/mL, LOQ was 7.385 µg/mL, accuracy was 100.68%, precision was 0.82% for neomycin sulfate. Levels of BTS was 111.1 ± 0.97% and levels for neomycin sulfate was 100.91 ± 0.37%. The proposed method has met ICH guidelines and can be used for simultaneous estimation of betamethasone valerate and neomycin sulfate in cream form.