The use of appropriately chosen reference genes for normalizing gene expression in real-time quantitative reverse transcription polymerase chain reaction is an important step in the analysis of gene expression, compensating for several technical factors. As female sex hormones have been shown to influence growth and differentiation of thyroid follicular cells, the establishment of normalizer genes in human thyroid cells in primary culture, treated with progesterone, and estradiol, is important to evaluate their effect on gene expression in these cells, so candidate reference genes were studied. β-Actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β2-microglobulin (B2M), and TATA box binding protein (TBP) were evaluated in thyroid cells treated with estradiol, progesterone, and their inhibitors. Normfinder software was used to assess the stability of the genes and identified β-actin as the gene with adequate stability and lower inter-group variations, when compared to TBP, B2M, and GAPDH.