Mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) is an inherited lysosomal storage disease caused by deficiency of Nacetylgalactosamine-6-sulfatase (GALNS), an enzyme required for stepwise degradation of keratan sulfate (KS). KS is an important biomarker for MPS IVA as it appears in the circulation and urine, and KS levels can be used to differentiate between MPS IVA patients and unaffected individuals. We have developed a selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay for the KS-derived disaccharides Galb1-4GlcNAc(6S) and Gal(6S)b1-4GlcNAc (6S) in human urine and plasma using keratanase II digestion. This method expands on the existing LC/MS/MS method by allowing quantitation in both urine and plasma and using improved internal standards (heavy isotope-labeled Galβ1-4GlcNAc(6S) and Gal(6S)b1-4GlcNAc(6S)). The lower limit of quantitation was 0.026 μg/mL (plasma) and 0.104 μg/mL (urine), with a quantitation range of 0.026-5 μg/mL (plasma) and 0.104-20 μg/mL (urine). Clinical sample analysis in 168 MPS IVA patients and 225 healthy controls demonstrates the clinical utility of this method in identifying reference ranges for KS in urine and plasma and discriminating between individuals with MPS IVA and unaffected individuals. It also has the potential for longitudinal disease progression assessment. Conflict of Interest declared.