Ornithine transcarbamylase (OTC) deficiency is an X-linked, semidominant genetic disorder and the most prevalent inherited defect of the urea cycle. Molecular genetic testing of the OTC gene is critically important for clinical diagnosis, carrier testing, and prenatal diagnosis. Private mutations are observed throughout the OTC gene with more than 340 reported disease-causing mutations. High-resolution melt profiling was adapted to perform homogeneous analysis of the 10 coding regions and their intronic flanks in a 96-well plate format. The 10 DNA fragments ranging from 146 bp to 266 bp are amplified in a PCR run. A common analysis condition simultaneously generates melting profiles from all 10 fragments. To streamline analysis, deviant profiles resulting from common polymorphic variants are triaged using redundant assessment with melt profile controls in selected whole-exon assays and a separate multiplex genotyping assay. The test is further streamlined by recovering dye-stained amplification product from the melt profiling plate to serve as DNA sequencing template. Described herein is the comprehensive analysis of the OTC gene in 23 OTC-deficient patients. This system provides a rapid means to localize sequence variants, markedly reducing the need for DNA sequencing, and is applicable to other genes and disorders.