microRNAs (miRNAs) are noncoding RNAs of approximately 24 nucleotides long that are expressed in multiple tissues and organs. miRNAs regulate the expression of specific target genes, and their expression profile can provide insight into the cell biology altered by diseases or drug treatments. Research has demonstrated that several miRNAs regulate muscle differentiation and the specification of fiber type. Hence, an established tool to routinely quantify miRNA abundance is of the utmost importance. The stem–loop quantitative reverse transcription PCR (RT-qPCR) is a methodology that allows the analysis of a specific miRNA with high sensitivity. Upon hybridization with a structured stem-loop oligonucleotide to the 3′ end of the target miRNA, a retro-transcription is possible for further qPCR quantification. In this chapter, we describe a protocol to quantify miRNAs in dissected mouse fast and slow-twitch muscle.