Determination of radiochemical purity is essential for characterization of all radioactive compounds, including clinical radiopharmaceuticals. Radio-thin layer chromatography (radio-TLC) has been used as the gold standard for measurement of radiochemical purity; however, this method has several limitations in terms of sensitivity, spatial resolution, two-dimensional scanning, and quantification accuracy. Here, we report a new analytical technique for determination of radiochemical purity based on Cerenkov luminescence imaging (CLI), whereby entire TLC plates are visualized by detection of Cerenkov radiation. Sixteen routinely used TLC plates were tested in combination with three different radioisotopes (131I, 124I, and 32P). All TLC plates doped with a fluorescent indicator showed excellent detection sensitivity with scanning times of less than 1 min. The new CLI method was superior to the traditional radio-TLC scanning method in terms of sensitivity, scanning time, spatial resolution, and two-dimensional scanning. The CLI method also showed better quantification features across a wider range of radioactivity values compared with radio-TLC and classical zonal analysis, especially for β--emitters such as 131I and 32P.