Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous molecule in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small molecules in live cells. An examination of structure Raman shift/intensity relationships revealed that alkynes conjugated to an aromatic ring and/or to a second allcyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular concentrations of diyne-tagged Coganalogues could be semiquantitatively estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2 '-deoxyuridine (EdU) and a Cog analogue, with distinct Raman tags was demonstrated.