Deficiency of β-Glucocerebrosidase (GBA) activity causes Gaucher Disease (GD). GD can be diagnosed by measuring GBA activity (Beutler and Kuhl, 1990). In this study, we assayed dried blood spots from a cohort (n=528) enriched for GBA mutation carriers (n=78) and GD patients (n=18) using both the tandem mass spectrometry (MS/MS) and fluorescence assays and their respective synthetic substrates. The MS/MS assay differentiated normal controls, which included GBA mutation carriers, from GD patients with no overlap. The fluorescence assay did not always differentiate normal controls including GBA mutation carriers from GD patients and false positives were observed. The MS/MS assay improved specificity compared to the fluorescence assay.