A new spectrofluorimetric method has been developed for the quantification of danofloxacin (DANO) and difloxacin (DIFLO), in the presence of the primary metabolite of difloxacin, with sarafloxacin (SARA) as interference, in chicken tissue samples. The method is based on second-order multivariate calibration, applying parallel factor analysis (PARAFAC), to the excitation-emission matrices (EEMs) of these compounds. High overlapping of the signals and influence of matrix effects were observed. To solve the problem, the standard addition method was used. Chemical variables were optimized. The measured EEMs of the analytes, as analytical signals, allowed their quantification in chicken tissue samples. Solid phase extraction was used for the extraction of the analytes in real samples. The range of concentration examined varied from 30 to 100 ng g(-1) for danofloxacin, and from 100 to 200 ng g(-1) for difloxacin. Both analytes can be analyzed individually, and the binary mixture can be resolved, with recoveries comprising between 88.7 and 106.6%.