Hypothyroidism is a pathological condition that occurs due to a decrease in the formation and secretion of thyroid hormones. Hypothyroidism therapy relies on the use of levothyroxine, but a subgroup of patients with residual symptoms has been identified despite this therapy. That is why in the last two decades an increasing number of patients are resorting to thyroid preparations that contain triiodothyronine in addition to thyroxine. Natural thyroid preparations are by composition dry powder of the thyroid gland. The problem of their use is unresolved issues of content that differs between manufacturers but also between individual series of the same manufacturer. A small number of studies have been done so far with the aim of solving this issue, so the aim of this paper is to develop and validate a fast, specific and reliable method of High-performance liquid chromatography on the reverse phase with UV-Vis detection for simultaneous quantification of T3 and T4 in such biological sample. Anthraquinone was used as an internal standard for quantifying T3 and T4. The method is validated by examining the accuracy, precision, specificity, linearity and limit of detection limit of quantification. The developed method is highly specific (RSD 0.41170 % for T3 and 0.35716 % for T4) and accurate (analytical yield 103.29 % - 116.81 %). The method is linear in the concentration area studied (0.125-2.5 μg/mL). Intra and inter-test accuracy is high (RSD for T3 and T4 and internal standard was lower than 5%). Limit of detection and limit od quatification are low for both analytes (LOD 80 ng/mL, LOQ 243 ng/mL). The method was applied for quantitative analysis of T3 and T4 in a commercial thyroid preparation inside and outside the shelf life. The results of quantitative analysis of the thyroid preparation that was within the shelf life correspond to the declared content values, while the sample analysis outside the shelf life showed a decreased amount of T3 and T4.