Second derivative spectrophotometry has been useful for the determination of aromatic amino acids. However, published methods produce erroneous results, because those methods measure second derivative values by the vertical distance between peak and trough which is subject to variation according to the aromatic amino acid composition of proteins. This paper presents a method of second derivative spectrophotometry which measures second derivative absorbance values by means of the vertical distance from baseline to the derivative curve at a wavelength specifically assigned to each aromatic amino acid, and makes corrections for the interference from other amino acids at the same wavelength. The Appendix describes a computational method for obtaining absolute values of second derivative absorbances directly from normal absorbance values without using the spectrophotometer's derivative mode, because most commercial instruments produce completely arbitrary second derivative values which make comparison of data obtained on two different instruments impossible.