BACKGROUND Urinary excretion of the tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc₄) is increased in various clinical conditions associated with increased turnover or storage of glycogen, making Glc₄ a potential biomarker for glycogen storage diseases (GSD). We developed an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to detect Glc₄ in urine without interference of the Glc₄ isomer maltotetraose (M₄). METHODS Urine samples, diluted in 0.1% ammonium hydroxide containing the internal standard acarbose, were filtered, and the filtrate was analyzed by UPLC-MS/MS. RESULTS We separated and quantified acarbose, M₄, and Glc₄ using the ion pairs m/z 644/161, 665/161, and 665/179, respectively. Response of Glc₄ was linear up to 1500 μmol/L and the limit of quantification was 2.8 μmol/L. Intra- and interassay CVs were 18.0% and 18.4% (10 μmol/L Glc₄), and 10.5% and 16.2% (200 μmol/L Glc₄). Glc₄ in control individuals (n = 116) decreased with increasing age from a mean value of 8.9 mmol/mol to 1.0 mmol/mol creatinine. M₄ was present in 5% of urine samples. Mean Glc₄ concentrations per age group in untreated patients with Pompe disease (GSD type II) (n = 66) were significantly higher, ranging from 39.4 to 10.3 mmol/mol creatinine (P < 0.001-0.005). The diagnostic sensitivity of Glc₄ for GSD-II was 98.5% and the diagnostic specificity 92%. Urine Glc₄ was also increased in GSD-III (8 of 9), GSD-IV (2 of 3) and GSD-IX (6 of 10) patients. CONCLUSIONS The UPLC-MS/MS assay of Glc₄ in urine was discriminative between Glc₄ and M₄ and confirmed the diagnosis in >98% of GSD-II cases.