DPYD testing is recommended by drug agencies prior starting treatment with fluoropyrimidines to avoid severe adverse reactions. However, its implementation in poor regions of Brasil is complicated because of cost and the technologies used. Our aim was to develop a new and inexpensive DPYD assay including relevant variants in Latin American population that avoid the use of fluorescency equipment. Specific oligonucleotides for a multiplex allele-specific PCR for 5 DPYD single nucleotide polymorphisms (SNPs) (c.1679T>G, c.1905+1G>A, c.2846A>T, c.1129.5923C>G and c.557A>G) were designed.