The chapter discusses the evolution and advancements in real-time quantitative PCR (qPCR) technology. Initially, early qPCR techniques were cumbersome and required careful management of linear amplification and product detection. However, real-time qPCR has revolutionized the field by offering faster data generation, enhanced sensitivity, high throughput, and reduced variability. The technology has seen a significant increase in usage over the past decade, with a notable rise in citations and technological advancements. This thematic issue highlights various techniques and applications of real-time qPCR, including cytokine gene expression quantification, absolute and relative quantification methods, validation of differential gene expression, DNA methylation patterns, allelic discrimination, and analysis of archival biopsies. Real-time qPCR has also improved high-throughput screening capabilities, such as the detection of single-nucleotide polymorphisms (SNPs) and multiplex PCR reactions. The chapter emphasizes the transformative impact of real-time qPCR on routine diagnostic assays and the ongoing development of associated technologies.The chapter discusses the evolution and advancements in real-time quantitative PCR (qPCR) technology. Initially, early qPCR techniques were cumbersome and required careful management of linear amplification and product detection. However, real-time qPCR has revolutionized the field by offering faster data generation, enhanced sensitivity, high throughput, and reduced variability. The technology has seen a significant increase in usage over the past decade, with a notable rise in citations and technological advancements. This thematic issue highlights various techniques and applications of real-time qPCR, including cytokine gene expression quantification, absolute and relative quantification methods, validation of differential gene expression, DNA methylation patterns, allelic discrimination, and analysis of archival biopsies. Real-time qPCR has also improved high-throughput screening capabilities, such as the detection of single-nucleotide polymorphisms (SNPs) and multiplex PCR reactions. The chapter emphasizes the transformative impact of real-time qPCR on routine diagnostic assays and the ongoing development of associated technologies.