A first-derivative spectrophotometric method was developed for quantitative determination of two synthetic organic dyes, Carmoisine and Patent Blue V, which are under governmental regulations all over the world because of their toxicity and carcinogenity. In this study, Carmoisine and Patent Blue V were simultaneously determined in their binary mixtures by first-derivative spectrophotometry. The method was applied to different gelatine desserts. The first-derivative derivative method did not eliminate turbid background in the samples. A simple background correction procedure involving a C-18-cartridge sample preparation step was developed for determination of dyes in gelatine dessert by first-derivative spectrophotometry. The C-18-cartridge sample preparation step improves recovery in determination of Carmoisine and Patent Blue V in gelatine dessert by first-spectrophotometry. Recoveries for Carmoisine and Patent Blue V in different samples were generally higher than 90%. This method also provides rapid, accurate and economical analysis of these dyes.