Autoradiography generally involves in metallic silver stain formation through irradiation to photosensitive materials (such as X-ray film) with further development and fixation steps using a radiolabeled probe, and the stain density indicates the relative amount of its target molecules and its distribution in tissue slices. This traditional method has a wide range of applications both in biological studies and preclinical drug researches. Phosphor film imaging has significantly shortened the autoradiography’s experimental intervals, and the positron nuclides with short-half-lives can also be performed. The purpose of this paper focuses on the frontier of some experimental works that can’t be completely replaced by non-radioactive labeled methods, such as some enzymatic activity assays, protein or peptide phosphorylation site analyses, nucleic acid assays with low concentrations, and the target (such as receptors) molecule distributions in tissue slices by radiolabeled selective ligands. Thus the technological platform should be reformulated to adapt to the current trends of experimental works.