<p>Ribosome profiling (Ribo-seq) is a next-generation, high-resolution sequencing technique that captures ribosome-protected mRNA fragments to map ribosome positions across the transcriptome. This method serves as a powerful proxy for global translational activity by revealing where ribosomes engage with mRNAs. Recent advances have expanded the utility of Ribo-seq to resolve distinct ribosome populations, including initiating ribosomes, small subunits, collided ribosomes, mitochondrial ribosomes, and those associated with specific translation factors or localized to subcellular compartments. These methodological advances have significantly broadened the scope of Ribo-seq, enabling new insights into the molecular mechanisms that govern translation across diverse eukaryotic systems. In this mini-review, we highlight key innovations in Ribo-seq technology and discuss how they have deepened our understanding of the spatial, temporal, and regulatory dimensions of translational control.</p>