|Thyroxine (T4) and Triiodothyronine (T3) are tyrosine-based hormones produced by the thyroid gland and are primarily responsible for the regulation of metabolism. More than 99% of T3 and T4 transported in blood is bound to transport proteins. The remaining is free and biologically active. An LC/MS/MS method was developed for the quantitative analysis of T3 and T4 using Multiple Reaction Monitoring (MRM) in ESI negative mode, the most sensitive and selective scan mode for quantitation. Based on this work, we evaluated the quantitative integrity of T3 and T4 in solvent as well as in matrix.