A method using UV-Vis spectroscopy and multivariate tools for simultaneous determination of glucose and cholesterol was developed in this paper. The method is based on the development of the reaction between the analytes (cholesterol and glucose) and enzymatic reagents. The spectra were analyzed by partial least squares regression and artificial neural networks. The precision estimated between nominal and calculate concentration demonstrate that artificial neural network model was adequate to quantify both analytes in serum samples, since the % relative error obtained was in the interval from 5.1 to 8.3. The proposed model was applied to analyze blood serum samples, and the results are similar compared to those obtained employing the reference method.