Global Run-On sequencing (GRO-seq) is one of the most sensitive techniques to detect nascent transcription from RNA polymerase (Pol) at a genome-wide level. The protocol incorporates labeled ribonucleotides into nascent RNAs from Pol I, II, and III. We have adapted the GRO-seq protocol to the nematode Caenorhabditis elegans to measure transcription from embryos and adult worms. Here, we provide a detailed overview of the protocol highlighting the critical steps for generating successful libraries. For complete details on the use and execution of this protocol, please refer to Quarato et al. (2021).