Ribosome profiling (or ribo-sequencing) is a powerful technique that enables high-resolution analysis of active translation by sequencing ribosome-protected mRNA fragments. Developed in 2009 by Nicholas Ingolia and Jonathan Weissman, it provides direct insights into which mRNAs are being translated and at what rate, surpassing traditional transcriptomic and proteomic methods. Ribo-seq allows for precise mapping of ribosome positions, enabling detailed characterization of ribosome dynamics and the identification of alternative translation initiation sites and upstream open reading frames. The standard workflow includes key steps such as ribosome stabilization, nuclease digestion, fragment isolation, and deep sequencing. The protocol described in this chapter incorporates a polysome profiling step prior to RNase treatment, allowing, for instance, the isolation of distinct ribosome species (also known as k-somes). Ribo-seq has transformed our understanding of translational regulation and has become an essential tool for omics studies in various fields such as developmental biology, cancer biology, virology, and microbiology.