The present invention belongs to the field of analytical chemistry, and relates to a method for separation and determination of two medicine contents and related substances in a tazarotene-betamethasone cream. According to the method, a reagent composition for solid-liquid separation and determination of two medicine contents and related substances in a tazarotene-betamethasone cream comprises an acidic aqueous solution system and acetonitrile, wherein the acidic aqueous solution system comprises an acidic additive, methanol and water; and by using the reagent composition, the two medicine contents and the related substances in the tazarotene-betamethasone cream can be simultaneously separated and determined through high performance liquid chromatography. According to the present invention, the method has good reproducibility, the solvent peak does not interfere with the determination of the two medicine contents and the related substances in the tazarotene-betamethasone cream during the detection, the two medicines and the related substances in the tazarotene-betamethasone cream can be effectively separated, the separation degree is more than 2.0, the detection result is accurate and reliable, and the important significance is provided for the achievement of the quality control of the tazarotene-betamethasone cream.