An analytical methodology was developed for qualitative and quantitative impurity profiling of the coloring agent Sudan III by high-performance liquid chromatography (HPLC)-diode array detection (DAD). The impurities in commercial Sudan III were characterized by comparison of their retention times and UV spectra with those of authentic standards. Four impurities regulated by International Committees in Sudan III were quantified by HPLC-selective UV detection. The impurities in Sudan dye were successfully separated on a reversed phase C18-column within 25 min and sensitively detected by UV spectrometry at two selective wavelengths. Method validation was conducted to determine linearity, precision, accuracy, and limit of quantification (LOQ). The linear dynamic range extended from 0.002 to 4.0%, with a correlation coefficient (R2) greater than 0.995. The LOQs of the impurities ranged from 8.04 to $54.29{mu}g/mg$. Based on the established method, the levels of regulated impurities in five commercial Sudan III dyes were determined.