Simple and novel spectrophotometric method is described for simultaneous determination of aluminum and iron. The method is based on the colored complexes formed by aluminum and iron with hematoxylin in the presence of cetyltrimethylammonium bromide as a surfactant at pH 5.8. All factors affecting the sensitivity were optimized and the linear dynamic range for determination of aluminum and iron was found. The simultaneous determination of aluminum and iron mixtures by using spectrophotometric methods is a difficult problem, due to spectral interferences. By multivariate calibration methods such as partial least squares (PLS), it is possible to obtain a model adjusted to the concentration values of the mixtures used in the calibration range. Orthogonal signal correction (OSC) is a preprocessing technique used for removing the unrelated information and is a suitable preprocessing method for PLS calibration of mixtures without loss of prediction capacity using spectrophotometric method. In this study, the calibration model is based on absorption spectra in the 360-620 nm range for 25 different mixtures of aluminum and iron. Calibration matrices contained 0.06-21.00 and 0.03-15.00 μg mL-1 of aluminum and iron, respectively. The RMSEP for aluminum and iron with OSC and without OSC were 0.06, 0.08 and 0.24, 0.27, respectively. The proposed method has been applied to the simultaneous determination of aluminum and iron in plant and water samples.