Transcriptome-wide translation can be measured using ribosome profiling, a technique which relies on deep sequencing of ribosome protected fragments. This method enables comprehensive measurement of in vivo translation; however, it involves multiple steps and thereby demands high-input samples. Here, we describe Ribo-ITP (RIBOsome profiling via IsoTachoPhoresis), which utilizes the principles of isotachophoresis coupled with a stringent size selection strategy in a microfluidic chip to isolate ribosome-protected fragments. Ribo-ITP allows quantitative measurement of translation from rare populations like cancer stem cells, early embryonic stages, and other ultralow input samples including single cells.