Lipid domains in supported lipid layers serve as a popular model to gain insight into the processes associated with the compartmentalization of biological membranes into so-called lipid rafts. In this paper, we present reproducible tip-enhanced Raman spectra originating from a very small number of molecules in a lipid monolayer on a gold surface, probed by the apex of a nanometer-sized silver tip. For the first time, we show large (128 × 128 pixels), high-resolution (< 50 nm) tip-enhanced Raman images of binary lipid mixtures with full spectral information at each pixel.