Sphingomyelinases are a group of hydrolases that cleave sphingomyelin, a common component of plasma membranes, to form ceramide and phosphocholine. Ceramide is a second messenger that is present in virtually all cell types and regulates a variety of cellular functions such as proliferation, differentiation, apoptosis, and inflammation response. Inhibition of sphingomyelinase activity to reduce ceramide concentrations has recently emerged as a potential therapeutic approach for several diseases including atherosclerosis, pathogen infections, inflammation, diabetes, and obesity. To effectively screen compound collections for the identification of new sphingomyelinase inhibitors, we have developed a high-throughput assay utilizing the natural substrate sphingomyelin in 1,536-well plate format. The assay has a signal-to-basal ratio of 6.1-fold in pH 5.0 buffer and 4.3-fold in pH 6.5 buffer, indicating a robust assay for compound library screening. A screen of ~300,000 compounds using this assay led to the identification of eight compounds as sphingomyelinase inhibitors (IC(50)s = 1.7 to 38.2 μM) that exhibited different activities between the natural substrate assay and profluorescence substrate assay. The results demonstrate the robustness and effectiveness of the natural substrate sphingomyelinase assay for screening sphingomyelinase inhibitors.