Thyroid hormones and their derivatives are structurally related to the non-essential amino acid tyrosine (4-hydroxyphenylalanine). However, there are physicochemical differences that make it difficult to apply an analytical method for their simultaneous detection. This work focused on the optimization of a method using liquid chromatography-electrospray ionization mass spectrometry to measure eight compounds related to levothyroxine (T4). In addition, the influence of the additives to the mobile phase, the solvents for liquid-liquid extraction and the influence of the hydrolysis of the conjugated analytes were studied. Optimization of MRM transitions and collision energy for analytes and capillary voltage, nebulizer gas pressure, nozzle voltage, sheath gas flow, sheath gas temperature, drying gas flow and drying gas temperature for ionization source was done. The recovery of analytes was studied using five solvents and six solvent systems to introduce them into the liquid-liquid extraction and matrix cleanup steps. Different additives to the mobile phase were evaluated as well as the effectiveness of enzymatic and chemical hydrolysis. The best MRM transitions and source parameters were settled in order to generate an optimized instrumental method. The addition of ammonium formate, ammonium fluoride, and acetic acid to the mobile phase showed no improvement in responses compared to classic 0.1% formic acid. The use of tert-butyl methyl ether: isopropanol (75: 25, V: V) showed a suitable recovery of analytes to perform a liquid-liquid extraction, and n-hexane might be an appropriate solvent if a cleanup step is needed. The stability of T3, T4 and thyronine, checked after the hydrolysis with extracts of E. coli and H. pomatia showed good results, contrary to chemical hydrolysis that showed a total degradation of T3 and T4.