Abstract. BACKGROUND: Gaucher disease (GD) is a lysosomal storage disorder characterized by a deficiency of the lysosomal acid -D-glucosidase (GBA). The aim of this studywas to develop an ultramicro-fluorometric assay based on the method of Chamoles et al. for determining GBA activity in dried blood spots on filter paper (DBS). METHODS: The assay used 3-mm diameter blood spot and 8 mmol/l of 4-methylumbelliferyl--D-glucoside as enzymatic substrate. The reaction occurred in plates incubated at 37◦C for 20 hours and the enzyme activity was expressed in mol hydrolysed substrate/l blood/h. The fluorescence of the enzyme productwas automatically measured in a fluorometer-photometer reader (SUMA Technology). RESULTS: The intra and inter-assay coefficients of variation were lower than 9 and 12%, respectively, and the recovery range was 97–109%.Three patients with GD were correctly diagnosed using the ultramicroassay. Healthy newborn DBS samples (n = 3003) from the National Neonatal Screening Program were analyzed, and the mean GBA activity was 5.7 mol/l blood/h. Our assay showed high Pearson (n = 26; r = 0.99) and concordance correlations (ρc = 0.99) with the traditional method described by Chamoles et al. CONCLUSIONS: The analytical performance characteristics of our ultramicro-fluorometric assay suggest that it can be used in the diagnosis of GD in newborns and adults.