We present a detailed protocol for ribosome profiling, an approach that we developed to make comprehensive and quantitative measurements of translation in yeast. In this technique, ribosome positions are determined from their nuclease footprint on their mRNA template and the footprints are quantified by deep sequencing. Ribosome profiling has already enabled highly reproducible measurements of translational control. Because this technique reports on the exact position of ribosomes, it also revealed the presence of ribosomes on upstream open reading frames and demonstrated that ribosome density was higher near the beginning of protein-coding genes. Here, we describe nuclease digestion conditions that produce uniform ~28 nucleotide (nt) protected fragments of mRNA templates that indicate the exact position of translating ribosomes. We also give a protocol for converting these RNA fragments into a DNA library that can be sequenced using the Illumina Genome Analyzer. Unbiased conversion of anonymous, small RNAs into a sequencing library is challenging, and we discuss standards that played a key role in optimizing library generation. Finally, we discuss how deep sequencing data can be used to quantify gene expression at the level of translation.