A covalently closed loop structure provides circular RNA (circRNA) with more stability than conventional RNAs in linear form, making circRNA an emerging tool in RNA therapeutics. The qualification and quantification of circRNA after production is critical for its design and effectiveness assessments, particularly when the following applications could be affected by byproduct RNAs. Despite PCR-based methods effectively detecting low-abundance circRNA, they are unsuitable for assessing uncircularized RNA in a mass production fraction to maintain quality control. Here, we present a straightforward protocol for evaluating uncircularized byproduct RNAs from circRNA production. This method enrolls the template-independent RNA polymerase activity to add adenine tails (polyA) to the 3' ends of a linear RNA, making it easy to distinguish trace byproducts or uncircularized RNA from a pool of mass circRNA products. With conventional linear RNA and RNase R-treated circRNA as the positive and negative controls, the purity of a circRNA preparation could be readily resolved. Regardless of circRNA production strategies, this protocol provides a reliable and practical way to ensure the consistent quality of homemade circRNAs or to recheck circRNA quality from commercial manufacturing.