Background and ObjectivesField 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.FindingsThe 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.ConclusionsTreatment 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 NoveltyThe 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.