Determination of RNA stability is one of the basic issues addressed in studies of RNA metabolism. In a standard approach used for RNA half-life measurement synthesis of RNA is inhibited and then the steady-state level of RNA is quantified and used for calculations. Here, we present an optimized protocol for mitochondrial RNA stability studies without perturbation of transcription and present results produced for the mitochondrial CytB messenger RNA. This method was originally described for nuclear transcripts and involves metabolic labeling of RNA with 4-thiouridine.