A method is described for measuring the concentrations of both glucose and glutamine in binary mixtures from near infrared (NIR) absorption spectra. Spectra are collected over the range from 5000-4000/cm (2.0-2.5 mu m) with a 1-mm optical path length. Glucose absorbance features at 4710, 4400, and 4300/cm and glutamine features at 4700, 4580, and 4390/cm provide the analytical information required for the measurement. Multivariate calibration models are generated by using partial least squares (PLS) regression alone and PLS regression combined with a preprocessing digital Fourier filtering step. The ideal number of PLS factors and spectral range are identified separately for each analyte. In addition, the optimum Fourier filter parameters are established for both compounds. The best overall analytical performance is obtained by combining Fourier filtering and PLS regression. Glucose measurements are established over the concentration range from 1.66-59.91 mM, with a standard error of prediction (SEP) of 0.32 mM and a mean percent error of 1.84%. Glutamine can be measured over the concentration range from 1.10-30.65 mM with a SEP of 0.75 mM and a mean percent error of 6.67%. These results demonstrate the analytical utility of NLR spectroscopy for monitoring glucose and glutamine levels in mammalian and insect cell cultures.