Batch-reactive crystallization of the two polymorphs of l-glutamic acid was studied using in-line Raman and ATR FTIR spectroscopy. It was observed that the barrier to the nucleation of the stable β-form was higher, and thus the occurrence of β-form nucleation requires a higher supersaturation level. The local supersaturation level inside the reactive crystallizer is significantly affected by the feeding manner of the reactant. When the reactant was fed to a poorly mixed zone, such as the surface of the liquid, a high local supersaturation level was generated near the feeding point. This high local supersaturation level drastically increased with the increase in the concentrations of the reactants. As a consequence, the fraction of the β-form increased with the increase in reactants concentrations. On the other hand, feeding the reactant to a well-mixed zone near the impeller can avoid the occurrence of high local supersaturation, and therefore the dependence of the polymorphic composition of the final product on the concentration of the reactants can be reduced. The information obtained from the spectroscopy leads to improved understanding of the precipitation process and offers great potential for process optimization and control of crystalline quality.