Tuberous Sclerosis Complex (TSC) is a hereditary syndrome of multi-organ hamartomas or benign tumorigenesis with autosomal dominant mode of transmission. The tumorigenesis in this syndrome was discovered for its etiology as perturbed function of the mechanistic or mammalian target of rapamycin (mTOR) pathway. The hyperactivated function of mTOR pathway caused by the loss of upstream negative regulators or heterotrimeric tumor suppressor complex (hamartin-tuberin-TBC1D7) was found to be the molecular pathogenesis in this syndrome. Hamartin-coding TSC1 and tuberin-coding TSC2 loci are therefore the candidates for identification of disease-causing variant in TSC patients. In addition to the clinical manifestations, the presence of mutation in the causative genes of TSC with confirmed pathogenicity can be considered as one criterion for TSC definite diagnosis. As the higher prevalence of mutations found in TSC2 than the one in TSC1, the molecular approach in this study was designed for detecting the variant in the coding sequence of TSC2 by using the nuclease assay to determine the existence of mismatched base pairing of the amplicon in a case of 63-year-old Thai male patient with sporadic but definite TSC. After screening the whole coding sequence of TSC2, the sequence analysis of the suspected amplicon by Sanger sequencing revealed a novel frameshift, single-nucleotide insertion (NM_00548.5:c.1572dupC) in exon 15, leading to premature termination of coding region (p.Asn525GlnfsTer64). This pathogenic frameshift mutation causes the extremely truncated tuberin protein (587 compared with 1,807 amino acid residues of the wild-type) with mutated C-terminal domains (residues 525-587). This finding additionally provides better understanding of the TSC genotype-phenotype correlation when compared with previous reports of TSC patient. The approach for variant screening in the whole TSC2 coding sequence used in this study is suggested to the routine laboratory service for molecular genetic analysis of TSC patients as another appropriate method of choice in practice.