Radiopharmaceuticals play an essential role in the non-invasive molecular assessment of normal and diseased tissues in the clinic. Numerous radiopharmaceuticals have been approved for diagnostic and therapeutic applications; even more are under active development. The process of bringing new radiopharmaceuticals to market requires selecting and thoroughly validating appropriate candidate molecules. This review summarizes the development of reliable high-resolution methods for radionuclide quantification and imaging with in vitro cancer models, including 2D cell cultures and 3D spheroids and organoids. While in vivo testing provides the most comprehensive assessment of a tracer, in vitro models have the advantage of higher throughput, lower cost, improved reproducibility, and shorter time-to-results. High-resolution imaging and radiobioassays enable the characterization of radiotracer distribution and uptake kinetics from the level of single cells to the 3D tumor models derived from a cohort of cancer patients. In addition, when used with well-established radiotracers, these methods can include clinically relevant imaging biomarkers in preclinical cellular and organoid studies for bridging the gap across in vitro models and clinical practice. We discuss how these techniques may evolve to create tremendous new opportunities for future research in the rapidly developing field of molecular imaging.