The development of genome-wide RNA profiling technologies greatly facilitates the global analysis of gene expression. However, such technologies alone could not distinguish the contribution to cellular RNA abundance by transcription versus decay. To overcome such limitation, metabolic labeling of newly synthesized RNA with 4-thiouridine (4sU) combined with genome-wide RNA profiling was used to in parallel measure RNA transcription and decay. Here, we describe the detailed protocol for using metabolic labeling with 4sU to separate newly synthesized RNA from the preexisting RNA in mammalian cells.