Thyroid hormone receptor alpha (THRα) is a nuclear hormone receptor that binds triiodothyronine (T3) and acts as an important transcription factor in development, metabolism, and reproduction. The coding gene, THRA, has two major splicing isoforms in mammals, THRA1 and THRA2, which encode THRα1 and THRα1, respectively. The better characterized isoform, THRα1, is a transcriptional stimulator of genes involved in cell metabolism and growth. The less well characterized isoform, THRα2, lacks the Ligand Binding Domain (LBD) and may act as an inhibitor of THRα1 activity. Thus, the ratio of THRα1 to THRα2 isoforms is critical for transcriptional regulation in various tissues and during development and may be abnormal in a number of thyroid hormone resistance syndromes. However, the complete characterization of the THRα isoform expression pattern in healthy human tissues, and especially the study of changes in the ratio of THRα1 to THRα2 in patient cell cultures has been hampered by the lack of suitable tools to detect the isoform-specific expression patterns. Therefore, we developed a plasmid splicing detector that allows the visualization and quantification of the differential expression of THRA1 and THRA2 splicing isoforms in living single cells during time-lapse and perturbation experiments. This tool enables experiments to further characterize the role of THRα2 and to perform high-throughput drug screening. Molecules that modify THRA splicing may be developed into drugs for the treatment of thyroid hormone resistance syndromes.