Depending on the scope of the research project, categories of single-nucleotide polymorphism (SNP) genotyping experiments range from low to medium to high throughput, with each approach differing widely in cost, platform, and efficiency. Medium-throughput genotyping is generally appropriate for assaying up to 36 markers in 384 individuals and is commonly used for fine-mapping chromosomal regions identified in genome scans. Multiplexing, which allows for simultaneous assessment of multiple SNPs, is an efficient, rapid, and economic way to augment medium-throughput genotyping output and is readily performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this chapter, we describe a technique for medium-throughput genotyping using the iPLEX® Gold assay available from Sequenom, Inc. (San Diego, CA). This multiplex SNP genotyping platform incorporates locus-specific PCR amplification of genomic DNA, followed by shrimp alkaline phosphatase treatment to inactivate unincorporated nucleotides, single-base primer extension using mass-modified terminators, and MALDI-TOF MS for allele-specific detection. This protocol utilizes proprietary enzymes, software and SpectroCHIP® Arrays that are pre-spotted with a MALDI matrix.