Measuring translation initiation rates is difficult. Gao et al. developed quantitative translation initiation sequencing (QTI-seq), a technique that captures translation initiation sites (TISs) at single-nucleotide resolution in cells and solid tissues, based on dissociating elongating ribosomes from transcripts while preserving the initiating ribosomes. The authors used QTI-seq to profile the effects of amino acid starvation on HEK293 cells and mouse embryonic fibroblasts (MEFs). Starvation-responsive transcripts often displayed multiple TISs, indicating a role for alternative TISs in translation control. In mouse livers, fasting elicited a different response to that in MEFs: the translation initiation of ribosome biogenesis transcripts was not repressed, and that of transcripts encoding components of the proteasome was enhanced. This indicates that a continuous supply of ribosomal proteins is needed in the liver during prolonged fasting, and that the proteasome contributes to cellular amino acid supplies.