Graves' disease (GD) and subacute thyroiditis (SAT) are important causes of thyrotoxicosis. The differentiation between these diseases is of great value because it will affect the management plan of either of them. The study aimed to assess the triiodothyronine/free thyroxine (T3/fT4) ratio as a criterion for the differentiation of hyperthyroidism due to GD and SAT. A retrospective study with database retrieval was conducted at Faiha Specialized Diabetes, Endocrine and Metabolism Center (FDEMC), Basrah, southern Iraq. Patients attending the center who presented with thyrotoxicosis due to GD and SAT from January 2010 to January 2024 were included in the analysis that was conducted from October 2023 to February 2024. For comparison between GD and SAT, the baseline thyroid-stimulating hormone (TSH), fT4 andT3were used to calculate the fT4 ratio (fT4 level (ng/dL)/1.7 ng/dL), T3 ratio (T3 level (ng/dL)/200 ng/dL), and T3/fT4 ratio (T3 level (ng/dL)/fT4 (ng/dL)). As compared to SAT, patients with GD had a significantly lower TSH and higher T3, T3 ratio, and T3/fT4 ratio. A T3/fT4 ratio with a cutoff equal to or more than 25 had 95% sensitivityand 18.1% specificity for GD with 94.4% positive predictive value. Raising the cutoff to equal or more than 100 results in the reduction of sensitivity to 32.7%but with 100% specificity and positive predictive value. The T3/fT4 ratio presents as a valuable diagnostic tool in differentiating GD from SAT, with potential applications in refining the diagnostic approach to hyperthyroidism.