Fluorescence spectroscopy is a powerful tool in cell biology and is particularly useful to monitor processes in living cells. Site-specific labeling of proteins is normally accomplished with genetic fusion of proteins with reporters such as GFP; however, such probes are large and can interfere with the functions of the protein to which they are fused and they are limited in their photochemical properties. Small molecule fluorophores have a wider range of photochemical characteristics and are less likely to perturb protein structure, but covalent incorporation of such molecules tends to be non-specific. Advances in the incorporation of unnatural amino acids into proteins allow for development of small molecule fluorescent probes that specifically label a single site in a single protein. Current fluorophore-unnatural amino acid reactive pairs require long reaction times or non-physiological conditions to form the protein-fluorophore conjugate. Ideally, the ligation reaction will proceed at a reasonable rate at neutral pH, low temperatures (37 °C or below) and non-cytotoxic concentrations of the fluorophore. Progress in our lab toward the development of cell-compatable chemoselective ligation partners will be presented.