A method for the simultaneous determination of 4-(indol-3-yl)butyric acid (IBA) and α-naphthalene acetic acid (NAA) in mixtures by first-derivative spectrofluorimetry has been developed. The method is based on their native fluorescence at an apparent pH of 8.0 in an aqueous-ethanol medium (50% v/v ethanol). IBA was measured at λem= 336 nm, and NAA at λem= 358 nm. The range of application is between 0.2 and 100.0 μgl−1for IBA and between 0.2 and 50.0 μgl−1for NAA. The accuracy and precision of the method are reported. The method was applied to the determination of IBA and NAA in synthetic mixtures and commercial formulations.