Thyroid function is commonly assessed in dogs through serum measurements of free thyroxine (FT4) and thyroid-stimulating hormone (TSH). The accurate measurement of thyroid hormones is critical. In particular, the measurement of free thyroxine (FT4) is important for the diagnosis and management of thyroid disorders. Most laboratories measure FT4 using direct analog immunoassay methods. The reliability of the results obtained using these methods has often been questioned. Fifty-nine dogs for which the veterinarian had requested the evaluation of thyroid hormones were included in this study. FT4 and TSH were measured using chemiluminescence enzyme immunoassay (CLEIA). Furthermore, FT4 was also measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) preceded by ultrafiltration. Comparing FT4 with the two different methods, the results demonstrate that LC-MS/MS, preceded by ultrafiltration, provides more accurate and specific FT4 measurements, particularly in dogs with altered thyroid hormone values. Although CLEIA is cost-effective and widely used for screening, LC-MS/MS is recommended for the more precise assessment of thyroid function.