Next-generation sequencing (NGS) has revolutionized clinical genotyping, providing high-resolution HLA genotyping with a low ambiguity rate. This study aimed to develop new NGS-based HLA genotyping (HLAaccuTest, NGeneBio, Seoul, KOREA) on the Illumina MiSeq platform and validate the clinical performance. The analytical performance of HLAaccuTest was validated for 11 loci comprising HLA-A, -B, -C, -DRB1/3/4/5, -DQA1, -DQB1, -DPA1, and -DPB1 using 157 reference samples. Among the 345 clinical samples, 180 were tested for performance evaluation and protocol optimization, and 165 were used in clinical trials in the validation phase for five loci, including HLA-A, -B, -C, -DRB1, and -DQB1. In addition, the improvement in the resolution of ambiguous alleles was also evaluated and compared with other NGS-based HLA genotyping for 18 reference samples, including five overlapping samples in analytical performance validation. All reference materials produced 100% concordant results for 11 HLA loci, 96.9% (2092 of 2160 HLA alleles) of the clinical samples were matched with the SBT results in the pre-validation phase. After the optimization phase, the clinical trials in the validation phase showed 99.7% (1645/1650 alleles) concordance with the complete resolution for 34 ambiguity results. The retesting of five discordant cases resolved all issues and yielded 100% concordant results with the SBT method. Additionally, for ambiguity using 18 reference materials with ambiguous alleles, about 30% of ambiguous alleles were more resolved than Trusight HLA v2. HLAaccuTest was successfully validated using a large volume of clinical samples and is fully applicable to the clinical laboratory.