Chondroitin sulfate (CS) is a glycosaminoglycan consisting of repeating uronic acid, Nacetylgalactosamine disaccharide units [(HexAβ/α(1-3) GalNAcβ (1-4)] n. CS chains are polydisperse with respect to chain length, sulfate content and glucuronic acid epimerization content, resulting in a distribution of glycoforms for a chain bound to any given serine residue. Usually, CS glycoforms exist, differing in sulfation position and uronic acid epimerization. This work introduces a novel LC/MS/MS platform for the quantification of mixtures of CS oligosaccharides. The CS polysaccharides were partially depolymerized and labeled with either the light (d 0 ) or heavy (d 4 ) form of 2-anthranilic acid (2-AA). Excess reagent was removed and mixtures of the CS standard (d 0 ) and unknown (d 4 ) were made. The CS mixture was subjected to size exclusion chromatography (SEC) with on-line electrospray ionization mass spectrometrometric detection in the negative ion mode. Tandem mass spectra were acquired and quantification of unknown samples within the mixture was made using relative ion abundances of specific diagnostic ions. High accuracy and precision of the glycomics platform were demonstrated using glycoform mixtures made from standard CS preparations. The CS glycoform analysis method was then applied to cartilage extract, versican, and several dermatan sulfate preparations. This work presents the first application of a glycomics platform for the quantification of CS oligosaccharide mixtures to obtain specific information on the positions of GalNAc sulfation and uronic acid epimerization.Glycomics is applied biology and chemistry that focuses on the structure and function of carbohydrates. Carbohydrates have vital functions in the body; in particular, glycosylation serves to diversify protein functions. Despite their abundance, much less information is known about carbohydrates, as compared to genes and proteins, largely due to the absence of a simple code that determines their structures. Mass spectrometry (MS), high performance liquid chromatography and other analytical tools have advanced rapidly to support the growth of biomolecular applications in the field of proteomics. However, technologies and methods to address the most challenging problems in glycomics, particularly with respect to glycoform quantitative measurements, remain undeveloped.Glycosaminoglycans (GAGs) are linear polysaccharides that consist of repeating disaccharide units that are attached to proteoglycan core proteins on adherent animal cell surfaces and in extracellular matrices (1-5). GAGs play significant roles in the modulation of cellular signals through interactions with proteins, including growth factors and growth factor receptors (6-8). They mediate cell-cell and cell-matrix interactions and are crucial to cell development and