Gene therapy holds great promise for the treatment of diverse diseases, but widespread implementation is hindered by difficulties in assessing the success of transfection. The development of noninvasive reporter techniques based on appropriate molecules and imaging modalities may help to assay gene expression. Fluorophenyl-β-D-galactopyranosides provide a novel class of NMR active molecules, which are highly responsive to the action of β-galactosidase (β-gal), the product of the lacZ gene. The reporter molecules are stable in solution and with respect to wild-type cells, but the enzyme causes liberation of the aglycon, a fluorophenol, accompanied by distinct color formation and a 19F NMR chemical shift of 5-10 ppm, depending on pH. Synthetic strategy, experimental methods, and molecular and 19F NMR characteristics are reported for a series of molecules in solution, blood, and tumor cells. This class of molecules presents a new strategy for assaying gene expression with a highly versatile molecular structural platform.