Application of a partial least squares algorithm (PLS-1) is proposed in this work for simultaneous spectrophotometric determination of calcium and magnesium in human serum. Two chromogenic agents, Calmagite [1-(1-hydroxy-4-methyl-2-phenylazo)-2-naphtol-4-sulfonic acid] ando-Cresolphtalein [3′,3″-bis{[bis-(carboxymethyl)amino]methyl}-5′5″-dimethylphenolphtalein] were used, which form color complexes with the two elements studied. As expected, significant interferences were observed for the individual methods (Ca-o-Cresolphtalein and Mg-Calmagite) in the presence of the second metal. However, applying a full-spectrum chemometric method PLS-1, it was possible to eliminate these interferences in the calibration step and satisfactory prediction results were obtained for calcium and magnesium in real serum samples. The proposed procedure was validated using Refererence Serum (171760 Boehringer Mannheim, Germany). The analytical results obtained for human serum were compared with the results of AAS determination.