In recent years, sphingolipids, a diverse class of glycolipids, and their regulatory enzymes have emerged as important players in human health and disease. This is exemplified by lysosomal β-glucocerebrosidase (GCase) which cleaves the glycoside bond of glucosylceramide. Homozygous carriers of mutations of its encoding gene (GBA) develop the lysosomal storage disease Gaucher’s disease, additionally heterozygous mutation carriers are at a drastically increased risk of developing the neurodegenerative disease Parkinson’s disease. How loss of function and alterations in the levels of its natural substrate, glucosylceramide (GlcCer), impacts the pathogenesis of its related diseases is still poorly understood. However, the relevance of glucosylceramide regulation to central nervous system health is strengthened by observations that an alternative degradative glycoside hydrolase, non-lysosomal glucosylceramidase (GBA2), has also been identified as being involved in several human neurodegenerative diseases. One of the limitations in the study of these glucosylceramide processing enzymes is the lack of selective and sensitive chemicals tools to study their function. Herein, we describe novel fluorescence quenched substrates to study GCase in tissue homogenates, live cells, and in fixed cells. Through these studies we have developed some of the most sensitive live cell substrates known. We demonstrate that these tools are quantitative and accurately report on enzymatic activity across a wide variety of cell types and instrumentation platforms. We also address one of the main limitations in the study of non-lysosomal glucosylceramidase – the lack of selective inhibitors to perturb its function. Because of homology to the natural substrate, existing inhibitors show cross inhibition not only with GCase but also the glycosyltransferase glucosylceramide synthase. This complicates delineation of the contributions that GBA2 has on the regulation of GlcCer. We address this problem by identifying a non-iminosugar based inhibitor that is more potent and more selective than the current standard use GBA2 inhibitor. These new chemical tools will enable a more thorough interrogation of the role that GlcCer and its regulation play not only in cellular biology but also human health and disease.