Rapid and accurate binary mixture resolution of tryptophan and tyrosine was performed. Differential-derivative spectrophotometry with a zero-crossing measurement technique was used for the quantitative determination of tryptophan and tyrosine in laboratory-prepared mixtures. Neither sample pretreatment nor separation were required. Linear calibration graphs of differential second derivative values were (at 222.4 and 217.9 nm for tryptophan and tyrosine , respectively) versus concentration in the ranges 0.1-20.0 and 1.0-50.0 µg ml_1, and the linearity was satisfactory (r= 0.9987and r= 0.9997), for tryptophan and tyrosine, respectively) were obtained .The relative standard deviations were found to be less than 1.03%, indicating reasonable repeatibility of method.The results obtained by the proposed method has been statistically compared by means of Student t-test and by the variance ratio Ftest where shows a good agreement.