Herein, we developed a new real-time PCR-based kit (U-TOPTM HL Genotyping Kit Ver2), comprising six variants from two auditory neuropathy spectrum disorder (ANSD) genes (OTOF and ATP1A3) and five variants from three SNHL genes (MPZL2, COCH, and TMC1), with a distinct auditory phenotype, making this the first genotyping kit dedicated to ANSD. Here, MPZL2 is linked to auditory neuropathy.