AbstractRNA molecules and their expression dynamics play essential roles in the establishment of complex cellular phenotypes and/or in the rapid cellular adaption to environmental changes. Accordingly, analyzing RNA expression remains an important step to understand the molecular basis controlling the formation of cellular phenotypes, cellular homeostasis or disease progression. Steady-state RNA levels in the cells are controlled by the sum of highly dynamic molecular processes contributing to RNA expression and can be classified in transcription, maturation and degradation. The main goal of analyzing RNA dynamics is to disentangle the individual contribution of these molecular processes to the life cycle of a given RNA under different physiological conditions. In the recent years, the use of nonradioactive nucleotide/nucleoside analogs and improved chemistry, in combination with time-dependent and high-throughput analysis, have greatly expanded our understanding of RNA metabolism across various cell types, organisms, and growth conditions.In this chapter, we describe a step-by-step protocol allowing pulse labeling of RNA with the nonradioactive nucleotide analog, 4-thiouracil, in the eukaryotic model organism Saccharomyces cerevisiae and the model archaeon Haloferax volcanii.