The slight structural differences between carbohydrate isomers pose considerable difficulties for their identification and quantification. Herein, a rapid method for the absolute quantification of glucose and fructose isomers using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS). A novel reagent, 4-trimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-(3-aminophenylboronic acid) (TMNTA), was synthesized and employed as a reactive matrix. This matrix significantly enhances the ionization efficiency of monosaccharides while avoiding matrix interferences. Glucose-