Gene expression analysis is the chief aspect of molecular biology research. It provides the insights into the regulatory mechanism which governs the cellular processes. RT-PCR is a powerful and versatile tool for measuring the levels of gene expression accuracy. The following abstract presents an overview of T-PCR the principle involved and the related applications. RT-PCR involves the formation of complementary DNA (DNA) using reverse transcriptase enzyme which is further accompanied by amplification of cDNA with the help of PCR. This process enables the researchers to analyze gene expression patterns, identification of differently expressed genes, moreover understand the cellular responses to various stimuli or conditions. This experimental study highlights the key steps involved for RT-PCR, which includes RNA extraction, reverse transcription, primer design, PC amplification and analysis of data methods such as relative as quantification. Moreover it further discusses the advantages and limitations of RT-PCR compared to other gene expression analysis techniques, kind microarrays and RNA sequencing. Furthermore, this study emphasizes the significance of RT-PCR in various fields such as disease research, development of drug and agricultural biotechnology, where precise measurement of gene expression is crucial for understanding patho-physiological process and developing therapeutic intervention. The application of RI-PCR in gene expression analysis has further revolutionized molecular biology researches and paves the way for advancement in personalized medicine and treatment strategies. This study further serves as a brief reference for the researchers, student and professional seeking to understand the principle and application of RT-PCR in unwinding the of gene expression regulation.