Regulation of translation is a key layer in the control of gene expression, playing a critical role in many biological and pathophysiological settings. In the last decade, methodological advances have provided comprehensive insights into cellular transcriptomes and proteomes, however, the systems-wide analysis of translation has remained an unsolved experimental challenge. The recent advent of ‘ribosome-profiling’ has provided the methodological basis for comprehensive transcriptome-wide analyses of translation at sub-codon resolution by the precise and high-throughput determination of ribosomal positions on mRNA. Its application in a variety of organisms yielded novel insights into protein synthesis and its dynamic regulation, significantly advancing our understanding of the post-transcriptional regulation of gene expression. This practical course will combine theoretical sessions with hands-on experimentation and computation emphasising the key steps of the protocol and computational quality control.