Quantitative Real-Time PCR (qPCR) has become an essential technique, widely employed in life sciences, scientific research, and medical applications. While it is mostly utilized in gene expression analysis and clinical diagnostics, its applications extend to mutation detection, genotyping, forensics, and quality and microbiological control relying, for instance, on the quantification of DNA from pathogens or genetically modified organisms (GMOs). Nonspecific detection methods, such as those using DNA-binding dyes like SYBR Green I, offer a degree of flexibility, but may have limitations in terms of the robustness required for routine techniques. In contrast, fluorescent probes significantly enhance the specificity and sensitivity of qPCR assays, making them ideal for demanding applications that require high accuracy and sensitivity. This chapter provides a comprehensive guide for the design of qPCR assays with fluorescent hydrolysis probes. It places particular emphasis on ensuring the specificity of primers and probes, which is crucial for generating reliable and accurate results. By detailing these critical steps, the chapter aims to equip researchers with the knowledge needed to understand the basics of qPCR and to design robust and efficient qPCR assays.