A method for generating a net analyte signal calibration model for use in detecting and/or quantifying the amount of an analyte in a test subject. The net analyte signal can be generated by providing a set of in vivo infrared spectra for a test subject during a period in which an analyte concentration is essentially constant; calculating an optimal subspace of spectra that at least substantially describes all non-analyte dependent spectral variance in the in vivo spectra; providing a pure component infrared spectrum for the analyte; and calculating a net analyte signal spectrum from a data set comprising the optimal subspace spectra and the pure analyte spectrum. The net analyte signal calibration model can be used, for example, in measuring the concentration of analyte in a test subject, and/or for evaluating the analytical significance of an in vivo multivariate calibration model.