Gaucher disease (GD) is due to deficiency of acid-β-glucosidase (ABG) and comprises a clinical spectrum with variable age of onset and severity. We evaluated a tandem mass spectrometry (MS/MS) method to measure ABG activity for high through-put screening.ABG activity was measured in 3.2mm punches from dry blood spots (DBS). Each punch was incubated for 21h with the substrate D-Glucosyl-β1-1′-N-dodecanoyl-D-erythro-sphingosine [C12-glucocerebroside (C 36 H 69 NO 8 )] and internal standard N-myristoyl-D-erythro-sphingosine [C14-ceramide (C 32 H 63 NO 3 )]. The product and internal standard were quantified using MS/MS.ABG activities in anonymized newborn screening samples from NY State were (mean) 22.0μmol/h/L±(SD) 13.8μmol/h/L (n=2088, median 19.9μmol/h/L, 95%CI 22.59–21.41μmol/h/L). The enzymatic activity in DBS from 10 treatment naïve adult Gaucher patients was less than 4.2μmol/h/L. ABG activity was stable for 3months at room temperature a 20% activity reduction was observed. Inter- and intra-run imprecisions were 8% and 13.7%, respectively. The limit of detection was 0.75μmol/h/L and limit of quantification was 1.25μmol/h/L.The measurement of ABG activities in DBS using MS/MS is suitable for high-throughput analysis of at-risk individuals and potentially for newborn screening for GD.