Glyoxylic acid has been determined in an organic mixture consisting of oxalic acid, glyoxylic acid, glycolic acid, acetic acid, glyoxal and ethylene glycol by a new spectrophotometric method based on its reaction with tryptophan in presence of 1 RSD : Relative standard deviation 2 LOD: limit of detection 3 LOQ: limit of quantitation ferric chloride and concentrated sulphuric acid. The new method doesn't require prior separation of glyoxylic acid from the mixture. The reaction between tryptophan and glyoxylic acid was found to be affected by reaction conditions. Good control of temperature and heat removal and correct reagent's amounts are critical for success of the new method. The method was found to be effective within the range 0.00557-0.028M of glyoxylic acid concentration. The limit of detection, limit of quantitation, standard deviation, relative standard deviation and recovery of the proposed method were found comparable with a selected HPLC reference method. Both methods displayed same precision and credibility. Reaction stoichiometry of tryptophan to glyoxylic acid was found to be 2:3, and the product is tryptophan blue dye. Although it is a dialdehyde, glyoxal didn't react with tryptophan according to Hopkins-Cole test.