Laminitic horses commonly suffer from an endocrine disease such as equine metabolic syndrome. Hyperinsulinemia is considered a key factor in the pathogenesis of laminitis. Since insulin also affects protein turnover in the body, the resting plasma amino acid concentrations of obese horses that were presented for a combined glucose insulin test (CGIT) were determined. In total, 25 obese horses and two lean horses with recurrent laminitis underwent a CGIT. Of these, five were not insulin dysregulated (obese), 14 were insulin dysregulated (ID), and eight were insulin-dysregulated and laminitic (IDL). Significant differences in the resting concentrations between obese and insulin dysregulated and laminitic (citrulline p = 0.038, obese: 73.001 ± 12.661 nmol/mL, IDL: 49.194 ± 15.486 nmol/mL; GABA p = 0.02, obese: 28.234 ± 3.885 nmol/mL, IDL: 16.697 ± 1.679 nmol/mL; methionine p = 0.018, obese: 28.691 ± 5.913 nmol/mL, IDL: 20.143 ± 3.09 nmol/mL) as well as between insulin dysregulated individuals with and without laminitis (GABA p < 0.001, ID: 28.169 ± 6.739 nmol/mL) regarding three amino acids were determined. This may be an interesting approach, especially for diagnostic testing and possibly also for the feed supplements of horses at risk of developing laminitis. However, further research, including a higher number of cases, is required