Evaluating the response of maize crops to different nitrogen fertilization rates is essential to ensure their agronomic, environmental, and economic efficiency. In this study, the spectral information of maize plants subjected to five distinct nitrogen fertilization strategies was analyzed. The fertilization strategies were based on the practices commonly used in maize fields in the study area, with the aim of ensuring the research findings’ applicability. The spectral reflectance was measured using a spectroradiometer covering the 350–2500 nm range, and the results enabled the identification of optimal spectral regions for monitoring plants’ nitrogen status, particularly in the visible and infrared ranges. A Principal Component Analysis (PCA) of the reflectance data revealed the key wavelengths most sensitive to the nitrogen availability: 555 nm and 720 nm during the vegetative stage and 680 nm during the reproductive stage. This information will support the development of drone-mounted multispectral sensor systems for large-scale monitoring, as well as the design of low-cost sensors for early nitrogen deficiency detection. Furthermore, the study demonstrated the feasibility of estimating the cornstalk nitrate content based on direct reflectance measurements of maize stems. The prediction model showed satisfactory performance, with a coefficient of determination (R²) of 0.845 and a root mean square error of prediction (RMSECV) of 2035.3 ppm, indicating its strong potential for predicting the NO₃-N concentrations in maize stems.