Background and Objectives Field experiments were conducted to assess the influence of in-season split application of nitrogen (N) on grain protein, protein digestibility, and amino acid dynamics in grain sorghum, in three different environments. Treatments included a zero N check and eight treatments with varying N application timings and amounts. Findings The five high N treatments had significantly greater grain protein content than all the lower N treatments. As the total protein increased, the kafirin portion of the protein increased, whereas the albumin-globulin levels decreased. Nitrogen treatment had a significant effect on 13 of the 18 amino acids. High N applications significantly increased amino acids on a protein basis including alanine, glutamic acid, leucine, proline, and phenylalanine. Conclusions Treatment N92 with N applied at 92 kg N ha−1 as split application at three different growth stages including planting, panicle initiation, and booting emerged as the optimum N treatment for increasing protein and amino acid concentration in grain sorghum. Significance and Novelty The ideal N application approach identified can be used to screen a wide range of sorghum hybrids under varying levels of irrigation and the same N treatments can be adopted by producers to enhance grain protein in sorghum.