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Summary: Micropeptides are emerging as important regulators of various cellular processes. Long non-coding RNAs (lncRNAs) serve as a source of micropeptide-encoding small reading frames. The techniques to detect micropeptides or translating lncRNAs, such as mass spectrometry and ribosome profiling, are sophisticated and expensive. Here, we present an easy and cost-effective protocol to screen for potential micropeptide-encoding lncRNAs by polysome profiling in suspension cell lines. When combined with quantitative PCR, this protocol facilitates the identification of a number of translating lncRNAs simultaneously.For complete details on the use and execution of this protocol, please refer to Sun et al. (2021).