We developed RiboLace, a novel method based on a new puromycin-containing molecule, for the isolation of active ribosomes and associate RNAs by means of an antibody-free and tag-free pull-down approach. We compared riboLace translation estimates with protein levels determined by proteomics in MCF7 cells. RiboLace can be easily and rapidly used to obtain a transcriptome-wide snapshot of active translation from eukaryotic in vitro and in vivo systems.