A simple and accurate spectrophotometric method for simultaneous determination was used in this research of liothyronine and thyroxine in human serum samples. The method created on the zerocrossing calculation for second and third derivative spectrophotometry. The calibration graphs are in the concentration spectrum, linear of 1.0 -17.0 µgmL -1 and 1-18 µgmL -1 for liothyronine and thyroxine successively. The recoveries range from 93.78 -102.68 % for T3 and 93.47 -103.25 % for T4 with relative standard deviation less than 4.28% and 4.50% in all in stance for T3 and T4 respectively.