Sialic acid is critically involved in carbohydrate–protein interactions that regulate processes like cell growth, differentiation, adhesion, immune response, fertilization, cancer development, and viral infection. To detail the molecular basis on which sialic acid-containing glycans (glycoconjugates) exert their biological functions, chemical synthesis will play a vital role. Our group has been addressing the chemical synthesis of gangliosides and their functionalized analogs to clarify the unknowns about ganglioside’s behavior in the live cell membrane. Robust chemical methods for α-sialylation and the conjugation of glycan with lipid allowed for the development of the fluorescently labeled ganglioside probes, which retain biophysical nature as lipid raft molecules. The single-molecule imaging of gangliosides revealed the specific interaction between gangliosides and lipid raft constituting a cluster of GPI-anchored protein CD59 and cholesterol. This observation showed the continual and dynamic exchange of gangliosides between lipid raft domain and the bulk domain, indicating that lipid raft domains are dynamic entities.