Estimating nitrogen (N) concentration in situ is fundamental for managing the fertilization of the sugarcane crop. The purpose of this work was to develop estimation models that explain how N varies over time as a function of three spectral data transformations in two stages (plant cane and first ratoon) under variable rates of N application. A randomized complete-block experimental design was applied, with four levels of N fertilization: 0, 80, 160, and 240 kg N ha−1. Six sampling events were carried out during the rapid growth stage, where the canopy reflectance spectra with a hyperspectral sensor were measured, and tissue samples for N determination in plant cane and first ratoon were taken, from 60 days after emergence (DAE) and 60 days after harvest (DAH), respectively, until days 210 DAE and 210 DAH. To build the models, partial least squares regression analysis was used and was trained by three transformations of the spectral data: (i) average reflectance spectrum (R), (ii) multiple scatter correction and Savitzky-Golay filter MSC-SG) reflectance spectrum, and (iii) calculated vegetation indices (VIs).