This paper provides a simple, reliable, and inexpensive spectrophotometric method for determination of glyoxylic acid in such reaction mixtures directly without the need of separation. The method depends on a colored chromogen product formation between glyoxylic acid and tryptophan when they react according to a reaction discovered in 1901 by Hopkins and Cole , see eq. . They suggested that one molecule of glyoxylic acid can react with two molecules of tryptophan to give a crimson-violet color that absorbs at 540-545 nm. This reaction ideally occurs in an anhydrous medium pledged by concentrated sulphuric acid .