Real-time PCR is based on the conventional principles of PCR and, with the simple shift of emphasis from the end-product to the whole course of the PCR process, has established itself as the most sensitive and specific quantitative PCR method. Real-time PCR is, like any other modern method in molecular genetics, expanding, with potential applications even in proteomics. With a variety of detection chemistries, an increasing number of platforms, multiple choices for analytical methods and the jargon emerging along with these developments, real-time PCR is facing the risk of becoming an intimidating method, especially for beginners. Real-time PCR provides the basics, explains how they are exploited to run a real-time PCR assay, how the assays are run and where these assays are informative in real life. It addresses the most practical aspects of the techniques with the emphasis on 'how to do it in the laboratory'. Keeping with the spirit of the Advanced Methods Series, most chapters provide an experimental protocol as an example of a specific assay.