Each intracellular organelle critically depends on maintaining its specific lipid composition that in turn contributes to the biophysical properties of the membrane. With our knowledge increasing about the organization of membranes with defined microdomains of different lipid compositions, questions arise regarding the molecular mechanisms that underlie the targeting to/segregation from microdomains of a given protein. In addition to specific lipid-transmembrane segment interactions as a basis for partitioning, the presence in a given microdomain may alter the conformation of proteins and, thus, the activity and availability for regulatory modifications. However, for most proteins, the specific lipid environment of transmembrane segments as well as its relevance to protein function and overall membrane organization are largely unknown. To help fill this gap, we have synthesized a novel photoactive sphingolipid precursor that, together with a precursor for phosphoglycerolipids and with photo-cholesterol, was investigated in vivo with regard to specific protein transmembrane span-lipid interactions. As a proof of principle, we show specific labeling of the ceramide transporter with the sphingolipid probe and describe specific in vivo interactions of lipids with caveolin-1, phosphatidylinositol transfer protein beta, and the mature form of nicastrin. This novel photolabile sphingolipid probe allows the detection of protein-sphingolipid interactions within the membrane bilayer of living cells.