Levothyroxine, a synthetic thyroxine analog, is used for the treatment of hypothyroidism. The laboratory has a central role in the diagnosis of hypothyroidism, monitoring of patients and dosing of replacement therapy. Preanalytical considerations in thyroid function tests include time and procedure of blood sampling, diet, physical activity, stress and circadian rhythm. Thyroid hormone concentrations are also affected by various medications such as furosemide, salicylates, heparin and certain conditions such as those accompanied by increased estrogen levels, neonatal age and hepatitis. This study aimed to examine the effects of taking levothyroxine therapy before blood sampling to define the exact procedure of patient preparation for thyroid function tests based on the findings. Wilcoxon's paired test was used to determine whether there was a statistically significant difference in concentrations. The study was conducted prospectively in the period from November 2020 to March 2021 in Sestre milosrdnice University Hospital Centre on a small sample of 20 adult volunteers. Blood samples for TSH, T4, T3, FT4 and FT3 assays were obtained immediately before and 2.5 hours after taking levothyroxine. Measurements of TSH, T4, T3, FT4 and FT3 were performed with the chemiluminescence method with microparticles on Abbott Architect i2000 automatic analyzer. Wilcoxon's paired test was used to determine whether there was a statistically significant difference in concentrations. The results of the study showed a statistically significant decrease in TSH concentration due to its circadian rhythm of secretion. Levothyroxine therapy led to a statistically significant increase in T4 and FT4 concentrations. On the other hand, levothyroxine therapy did not cause a statistically significant change in T3 or FT3 concentration. Levothyroxine therapy has a clinically significant effect on thyroid function tests, more specifically on T4 and FT4 concentrations, so it is justified to require patients not to take therapy before blood sampling, but to do so after blood samples are obtained.