We developed a 50 SNP-multiplex assay for detection on a MALDI-TOF MS platform based on the SNPs in the 52 SNP-multiplex assay recently developed by the SNPforID Consortium. After PCR amplification, the products were purified on Qiagen columns and used as templates in one single base extension (SBE) multiplex reaction. Two different strategies were used to design the SBE multiplex: (1) Small 5'-tags (3-8ánt) that increased the masses of the SBE primers without changing the annealing temperature; (2) Cleavable primers with one RNA nucleotide which was later cleaved by a mixture of RNases. The SBE primers were extended with biotin labelled ddNTPs and purified on avidin beads ensuring that only the extended SBE primers were isolated and spotted on the MALDI-TOF anchor target. Detection of the 50 extended primers from the SBE reaction was performed in a mass range between 3000 and 10,000 m/z