Real-time quantitative PCR is a technique used to monitor the PCR reaction in real time. RT-qPCR is broadly classified into two types based on its purpose: absolute and relative quantification. Absolute quantification is used in a wide array of fields such as microbiology, food technology, and biotechnology to quantify the microbiological load/adulterants in a commodity/copy numbers respectively, whereas Relative quantification is used in the field of genomics and functional transcriptomics to perform gene expression analysis in biological experiments. A laboratory work that covers the basic principles involved in RT-qPCR and data analysis using the manual as well as the software methods are incorporated. The laboratory experiment was designed to provide insights on certain important principles such as primer characteristics, PCR efficiency, and melt curve analysis. This laboratory exercise provides all the significant components of RT-qPCR, which can be useful while performing an experiment in an undergraduate and graduate laboratory.