Since genomic data are widely available, many strategies have been implemented to reveal the function of specific nucleotides or amino acids in promoter regions or proteins, respectively. One of the methods most commonly used to determine the impact of mutations is the site-directed mutagenesis using the polymerase chain reaction (PCR). There are different published protocols to develop single or multiple site-directed mutagenesis. In this chapter, we reviewed the enzymes commonly used in site-directed mutagenesis, the methods for simple and multiple site-directed mutagenesis in large constructs, mediated by insertion of restriction sites. Other methods reviewed include high-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips, and those based on multi-site-directed mutagenesis, based on recombination. Software tools to design site-directed mutagenesis primers are also presented.