Lipids in biological membranes are asymmetrically distributed across the bilayer. The choline-containing lipids, phosphatidylcholine (PtdCho) and sphingomyelin (SM), are more abundant in the external leaflet. In contrast, the amino-containing glycerophospholipids, phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEth), are located preferentially on the cytoplasmic leaflet. The maintenance of transbilayer lipid asymmetry is essential for normal membrane function, and disruption of this asymmetry is associated with cell activation or pathological condition. The physiological role of ceramide formation in response to cell stimulation remains controversial. Ceramide formation serves many different functions at various locations in the cell. Despite the limited capacity for spontaneous intracellular diffusion or membrane flip-flop of lipids in membranes, we have found that ceramide production, via sphingomyelinase action or addition of external ceramide, induces the transbilayer lipid motion of the lipids within the cellular membrane. This chapter outlines various commonly used assays for measuring lipid flip-flop induced by ceramide in cell and model membranes.