Calibration is the first step in the prediction of concentrations from spectral measurements of chemical reaction systems. It is a well-known fact that the species in the calibration set must include those in the new set. Typically, the calibration set is constructed from nonreacting mixtures of known concentrations. In this paper, it is proposed instead to use the calibration data from reacting mixtures, thereby avoiding the independent variation of possibly highly-reactive intermediates. However, for the prediction to be correct, restrictions on the initial and inlet concentrations of the new data set must be imposed. When these restrictions cannot be met, calibration of data in reaction-variant form is proposed. The methodology is illustrated experimentally using an esterification reaction.