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Publisher Summary Many researches involve the analysis of multiple regions within a gene (e.g., CFTR) or the analysis of multiple loci (e.g., for human identification). Rather than perform individual polymerase chain reaction (PCR) amplification reactions for each region or locus, it is often desirable to amplify all sequences of interest simultaneously in a "multiplex" reaction. This chapter presents the optimization guidelines for multiplex PCR. There are various advantages of using multiplex PCR. It offers a significant time and cost saving benefit, especially when a large number of individuals need to be analyzed. Also, only a single aliquot of DNA or RNA is required rather than an aliquot for each marker to be analyzed. This aspect is vital for prenatal diagnosis, forensic applications, and some clinical applications in which tissue and DNA samples are repeatedly reduced. Reducing the number of tubes to which aliquots of DNA need to be added also minimizes the possibility of contamination and sample mix-up during reaction setup. It outlines the optimization strategies of multiplex PCR. Most of the guidelines and strategies used to optimize single-target PCR are similarly helpful for optimization of multiplex PCR systems. However, certain other critical factors need to be considered that are discussed in this chapter. Finally, the chapter also presents several strategies that should expedite the development of reliable multiplex amplification reactions.