The quantitative predictive abilities of derivative spectral analysis by using the zero-crossing point method were compared with the results obtained by the use of multivariate calibration methods. Mixtures of two pesticides, diuron and chlorpyrifos, were resolved by application of derivative spectrophotometry. Partial least squares (PLS-1 and PLS-2) and principal component regression (PCR), full spectrum calibration methods, were also applied, with previous optimization of the calibration matrix, using the absorption spectra and the first-derivative spectra. Both approaches were satisfactorily applied to the simultaneous determination of diuron and chlorpyrifos in synthetic mixtures. However, significant advantages were found in the determination of these pesticides in groundwater samples, previous solid-phase extraction with C18cartridges, by application of different chemometric approaches when the calibration matrix was performed using the first derivative of the absorption spectra.