Nitrogen (N) is one of the key nutrients supplied in agricultural production worldwide. Over-fertilization can have negative influences on the field and the regional level (e.g., agro-ecosystems). Remote sensing of the plant N of field crops presents a valuable tool for the monitoring of N flows in agro-ecosystems. Available data for validation of satellite-based remote sensing of N is scarce. Therefore, in this study, field spectrometer measurements were used to simulate data of the Sentinel-2 (S2) satellites developed for vegetation monitoring by the ESA. The prediction performance of normalized ratio indices (NRIs), random forest regression (RFR) and Gaussian processes regression (GPR) for plant-N-related traits was assessed on a diverse real-world dataset including multiple crops, field sites and years. The plant N traits included the mass-based N measure, N concentration in the biomass (Nconc), and an area-based N measure approximating the plant N uptake (NUP). Spectral indices such as normalized ratio indices (NRIs) performed well, but the RFR and GPR methods outperformed the NRIs. Key spectral bands for each trait were identified using the RFR variable importance measure and the Gaussian processes regression band analysis tool (GPR-BAT), highlighting the importance of the short-wave infrared (SWIR) region for estimation of plant Nconc—and to a lesser extent the NUP. The red edge (RE) region was also important. The GPR-BAT showed that five bands were sufficient for plant N trait and leaf area index (LAI) estimation and that a surplus of bands effectively reduced prediction performance. A global sensitivity analysis (GSA) was performed on all traits simultaneously, showing the dominance of the LAI in the mixed remote sensing signal. To delineate the plant-N-related traits from this signal, regional and/or national data collection campaigns producing large crop spectral libraries (CSL) are needed. An improved database will likely enable the mapping of N at the agro-ecosystem level or for use in precision farming by farmers in the future.