A simple and accurate spectrophotometric method for simultaneous determination was used in this research of 3,3′,5-triidothyronine and thyroxine in human serum samples. The method created on the zero – crossing 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 T3 and T4 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.