MALDI-TOF is a simple and robust method for the analysis of single nucleotide polymorphism (SNP), which combines proven high-fidelity enzymatic procedures and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. This technology is among the most promising SNP typing methods for delivering accurate, effective, flexible and high throughput analysis of SNPs. “4-plex” genotyping protocol, which could detect 4 SNPs in one reaction, was performed in Beijing Genomics Institute with accuracy of 99.55%. To learn the correlation between genotyping call rates and quality, nearly ten thousands of typing results (“4-plex”) were analyzed. Significant positive correlation between call rate and the counts of conservative results were observed. SNPs with call rate lower than 82% have less conservative results, suggesting 82% was a cutoff to evaluate the genotyping results of MALDI-TOF assay. Aiming to further improve genotyping throughput and reduce the cost, “Mix 8-plex” and “Double-spotting 8-plex” genotyping protocols were developed. For new protocols, PCR and primer extension reactions were still performed under reliable “4-plex” protocol. For “Mix 8-plex” protocol, two sets of “4-plex” primer extension products were mixed and dispensed onto one SpectroCHIP at the same time. As to “Double-spotting 8-plex” protocol, two sets of “4-plex” products were consecutively dispensed onto one SpectroCHIP. 32 SNPs were genotyped in 95 human DNA samples to test the feasibility of these new protocols. The results showed that the performance of “Mix 8-plex” was as good as “4-plex” protocol, while the performance of “Double-spotting 8-plex” was poor.