Infection with high-risk (HR) human papillomaviruses (HPVs) has been confirmed as the necessary cause of cervical cancer. There are many studies that have established and confirmed the relationship of specific HPV types and the risk of invasive cervical cancer. We have developed a novel genotyping method for detecting 14 HR-HPV genotypes simultaneously with MassARRAY (Sequenom, San Diego, CA) technique based on the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). All 14 HPVs showed high specificities and high sensitivities in the plasmid test; lower detection limits for each genotype were from 10 to 100 copies. Furthermore, the MS system has high-throughput capacities, capable of processing, with type-specific output, 4,500 samples in 24 hours. The MS HPV assay is a sensitive and useful tool for HPV genotyping. It has the potential to be suitable for large-scale epidemiologic studies and routine diagnostic clinical applications owing to its high-throughput capacity, high sensitivity, and low cost per case.