Changes in neutrophil-to-lymphocite ratio (NLR) and platelet-to-lymphocyte ratio (PLR) have been identified in dogs with hypercortisolism (HC), but, no studies have investigated the changes in these inflammatory biomarkers as cost-effective and available parameters for the diagnosis and management of HC. This study was performed to evaluate whether NLR and PLR could be used as biomarkers for the diagnosis and treatment response in dogs with HC. This retrospective study included 67 dogs with HC, 58 dogs with non-adrenal illness (NAI), and 39 healthy dogs. NLR and PLR were compared among the three groups. Cut-off values of NLR and PLR for HC screening and percent change in biomarkers for assessing treatment response were evaluated. In addition, the NLR and PLR were compared before and after trilostane treatment. NLR and PLR were significantly higher in the HC group than in the NAI and healthy groups. The NLR cut-off value of 4.227 had a sensitivity of 67.16% and specificity of 65.52%, and the PLR cut-off value of 285.0 had a sensitivity of 56.72% and specificity of 70.69% for differentiating between dogs with HC and those with NAI, respectively. Furthermore, a significant decline in NLR was observed after treatment in the well-controlled HC group. The cutoff value of percent change in NLR to identify well-controlled HC was -7.570%; sensitivity and specificity were 100% and 63.64%, respectively. Therefore, NLR and PLR might be used cautiously as supportive biomarkers for HC diagnosis, and NLR could be a potential monitoring tool in assessing the treatment response of HC in dogs.