Quantitative study of protein requires accurate determination of protein concentration. Yet without a reliable value of protein extinction coefficient, it is difficult or impossible to determine protein concentration by the usual UV spectroscopic means. On the basis of the extinction coefficients of tyrosine and tryptophan in 0.1 mol/L NaOH solution, a method was introduced for determining accurate extinction coefficients for proteins at 280 nm by protein alkaline hydrolysis. Meanwhile, this method is calibrated against several proteins whose extinction coefficients are known from references. The results show that the extinction coefficients of these proteins determined by the alkaline hydrolysis method are accurate.