Derivative variable-angle synchronous fluorescence spectrometry is developed to improve the selectivity of fluorescence measurements without loss of sensitivity. The first derivative variable-angle synchronous scanning permits the rapid simultaneous determination of p-aminobenzoic acid, salicylic acid and gentisic acid in a mixture from a single spectrum based on a single scan. The analyses were performed in aqueous medium at pH 7 provided by the addition of phosphate buffer solution. A complete and exhaustive statistical analysis of the experimental data was realized to demonstrate the validity of the method.