Rheumatoid arthritis (RA) is an autoimmune inflammatory disorder associated with redness, warmth, swelling and acute pain within joint linings. Current means of diagnosing RA involve blood test, x-rays and MRI. Any combinations of these tests, however, fail to live up to consistent accuracy when diagnosing RA, potentially resulting in inadequate or improper therapies administrated by health care professionals. In order to find better means of diagnosing RA, research showed that the release of glycosaminoglycans (GAGs), heterogeneous and linear polysaccharides consisting of disaccharide repeat units, occurred during inflammation and infection. In order to better understand the role GAGs have in RA, we used an LC-MS/MS platform to analyze GAGs derived from healthy and RA infected human sera. We established mass spectrometry methods to quantify GAGs and obtained specific information on their sulfation and/or acetylation patterning at a hexuronic acid and hexosamine. Our investigation discovered statistically significant difference in the sulfation and acetylation patterns between RA and healthy sera. Furthermore, since isomers of the disaccharide exist, we established a MS method that generated diagnostic ions capable of distinguishing the eight additional disaccharide isomers. This mass spectrometry method may further increase the overall accuracy of RA diagnoses.