Fatty Acid Luciferin (FA-SS-luc) is a novel molecular imaging probe for a real-time noninvasive bioluminescent imaging of fatty uptake in cell culture and living animals. The probe design is based on a D-luciferin molecule conjugated to a long-chain fatty acid (palmitic, C16) via a cleavable disulfide bond that is stable in the extracellular environment but readily reduces intracellularly by glutathione after lipid uptake. The resulting free D-luciferin is oxidized by luciferase enzyme resulting in production of light proportional to the uptake of FA. Thus, this probe provides noninvasive, real-time, spatiotemporal and quantitative imaging of fatty acid uptake which opens a wide range of opportunities for establishing new drug screens and understanding fundamental biological processes involving fatty acid absorption both in vitro and in vivo.