Background: Graves’ disease and subacute thyroiditis are common causes of hyperthyroidism that share overlapping clinical features, making differentiation challenging. Accurate diagnosis is crucial for appropriate management to prevent adverse outcomes.Objective: To differentiate Graves’ disease from subacute thyroiditis using the ratio of serum free triiodothyronine (FT3) to free thyroxine (FT4).Methods: This prospective observational study was conducted at the Department of Diabetes, Endocrinology, and Metabolic Diseases, Hayatabad Medical Complex, Peshawar, from November 2023 to May 2024. A total of 248 patients diagnosed with Graves’ disease (n = 158) or subacute thyroiditis (n = 90) were included. Clinical, biochemical, and imaging data were collected, and the FT3/FT4 ratio was calculated. Data were analyzed using SPSS v25, with independent samples t-tests and ROC curve analysis performed.Results: The mean FT3/FT4 ratio was significantly higher in Graves’ disease (4.5 ± 0.8) compared to subacute thyroiditis (2.1 ± 0.5), p = 0.001. An FT3/FT4 ratio >3.0 had a sensitivity of 87% and specificity of 85%, with an AUC of 0.92.Conclusion: The FT3/FT4 ratio is a reliable diagnostic tool for differentiating Graves’ disease from subacute thyroiditis, offering high sensitivity and specificity.