Small open reading frame (smORF)-encoded microproteins, proteins containing less than 100-150 amino acids, are an emerging class of functional biomolecules. Here, we present a protocol for identifying translated smORFs in mammalian systems genome wide. We describe steps for generation of ribosome profiling (Ribo-seq) data, in silico translation of a transcriptome assembly to create an ORF database, and computational analysis of Ribo-seq to score individual smORFs for translation. Identification of translated smORFs is the first step to studying the functions of microproteins. For complete details on the use and execution of this protocol, please refer to Martinez et al.1.