Three-way fluorescence data and multivariate calibration based on parallel factor analysis (PARAFAC) are combined for the simultaneous quantitation of three fluoroquinolone anitibiotics (norfloxacin, enoxacin, and ofloxacin) in human serum samples. The three analytes can be adequately determined with limits of detection of 0.2, 3.0, and 0.5 microg L(-1), respectively, with minimum experimental effort. The selected analytical methodology fully exploits the so-called second-order advantage of the employed three-way data, allowing obtaining individual concentrations of calibrated analytes in the presence of any number of uncalibrated (serum) components. In contrast to PARAFAC, less satisfactory results were obtained with a multidimensional partial least-squares (nPLS) model trained with the same calibration set.