Grain protein content (GPC) is a key quality attribute and an important marketing trait in wheat. In the current cropping systems worldwide, GPC is mostly determined by nitrogen (N) fertilizer application. The objectives of this study were to understand the differences in N response between high and low GPC wheat genotypes, and to assess the value of biomass growth analysis to assess the differences in N response. Six wheat genotypes from a range of high to low GPC were grown in low, medium and high N, under glasshouse conditions. This experiment was designed around non-destructive estimation of biomass using a high throughput image-based phenotyping system. Results showed that Spitfire and Mace had higher grain N% than Gazelle and QAL2000, and appeared to demand more N to grow their biomass. Moreover, at low N, Spitfire grew faster and achieved the maximum absolute growth rate earlier than high N-treated plants. High grain N% genotypes seem able to manage grain N reserves by compromising biomass production at low N. This study also indicated the importance of biomass growth analysis to show the differences in the N responsiveness of high and low GPC wheat.