Although sphingomyelin and cholesterol are major lipids of mammalian cells, the detailed distribution of these lipids in cellular membranes remains still obscure. However, the recent development of protein probes that specifically bind sphingomyelin and/or cholesterol provides new information about the landscape of the lipid domains that are enriched with sphingomyelin or cholesterol or both. Here, we critically summarize the tools to study distribution and dynamics of sphingomyelin and cholesterol. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. Display omitted •Small molecules and proteins that bind specific lipids are employed as lipid probes.•Sphingomyelin (SM) and cholesterol (Chol) form characteristic lipid domains.•Lipid probes reveal the distribution of endogenous SM-and Chol-rich domains.•Fluorescent analogs of SM and Chol are useful to study dynamics of these lipids.