We explored the potential of iodine attachment to improve the sensitivity of glucose measurement by LC/MS. After sample preparation, glucose was separated by normal phase chromatography, followed by anionization by I(-)-attachment prior to MS by post-column addition of a methanolic solution of iodoform. Iodine is capable of forming an anionic adduct with neutral monosaccharides in negative ion mode electrospray mass spectrometry. Quasi-molecular ions [M+I]- of glucose, and [6,6-(2)H2]glucose (abbreviated d2-glucose) internal standard were quantitated in selected ion monitoring (SIM) mode. Iodine attachment LC/MS analysis provided high sensitivity, superior to GC/MS. It greatly simplified sample preparation and increased throughput. The advantages of iodine attachment can be realized even on old mass spectrometers. A LOD of 50 pg glucose on column was achieved. Due to iodine's predisposition to sublimate, the iodoform concentration must be minimized, which adds complexity to method development. To optimize reagent concentration we developed an efficient and flexible gradient-based delivery platform. Strategy for method development with iodoform is given.