RNA quantification is crucial for understanding gene expression and regulation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a widely used technique for RNA quantification because of its practical and quantitative nature, sensitivity, and specificity. Here, we provide an overview of RT-qPCR, focusing on essential reagents, the importance of primer design, the detailed workflow, and data analysis methods. This guide will be useful for scientists who are unfamiliar with RT-qPCR, highlighting key considerations for successful RNA quantification.