Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with a prevalence of 1 in 95,136 in Taiwan. TSC is characterized by hamartomatous lesions in multiple organ systems. Genetic defects in TSC1 and TSC2 genes are the main causes of TSC. A molecular screening protocol using denaturing high-performance liquid chromatography (dHPLC) followed by DNA sequencing is currently performed to locate the genetic lesions in many clinical laboratories. The current screening approach is time consuming and inefficient. In this study, we analyzed all coding exons of TSC1 and TSC2 genes of 30 TSC patients and 47 unaffected family members using the traditional dHPLC protocol and our recently developed diagnostic platform based on high-resolution melting analysis (HRM) followed by bidirectional DNA sequencing. Data indicated that 20 mutations, including 5 mutations in TSC1 (2 sporadic, 1 familial mutation, and 2 of uncertain origin) and 15 mutations in TSC2 (14 sporadic and 1 familial mutation), 8 single-nucleotide polymorphisms (SNPs, including 3 SNPs found in irrelevant individuals without TSC phenotypes studied in the control group), and 3 variants with undetermined significance were identified, including 4 novel mutations. The sensitivities of HRM and dHPLC for TSC mutation screening were estimated as 95% and 75%, respectively. The specificities of HRM and dHPLC for TSC mutation screening were evaluated as 91% and 98%. In addition, results suggested our novel HRM screening protocol to be more economical. In conclusion, we successfully developed a superior approach for TSC genes mutation screening for clinical application.