Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.