Thin-layer chromatography (TLC) is commonly employed to screen technetium-99m labeled polymer nanoparticle batches for unreduced pertechnetate and radio-colloidal impurities. Although this method is widely accepted, our findings applying radiolabeled PLGA/PLA–PEG nanoparticles underscore its lack of transferability between different settings and its limitations as a standalone quality control tool. While TLC profiles may appear similar for purified and radiocolloid containing nanoparticle formulations, their in vivo behavior can vary significantly, as demonstrated by discrepancies between TLC results and single-photon emission computed tomography (SPECT) and biodistribution data. This highlights the urgent need for a case-by-case evaluation of TLC methods for each specific nanoparticle type. Our study revealed that polymeric nanoparticles cannot be considered analytically uniform entities in the context of TLC analysis, emphasizing the complex interplay between nanoparticle composition, radiolabeling conditions, and subsequent biological behavior.