: Autism spectrum disorder (ASD) is a neurodevelopmental disorder that belong to genetic and epigenetic mechanism. Despite the recent advantages in next-generation sequencing (NGS) technology, ASD etiology is still unclear. : In this study, we tested a customized target genetic panel consisting of 74 genes in a cohort of 53 ASD individuals. The tested panel was designed from the SFARI database. : Among 53 patients analyzed using a targeted genetic panel, 102 rare variants were identified, with nine individuals carrying likely pathogenic or pathogenic variants considered genetically "positive." We identified six de novo variants across five genes ( 2 variants, , , , and ), including two variants of uncertain significance in p.Thr451Met and p.Glu1137Lys, one likely pathogenic variant in p.Leu714Gln, and three pathogenic variants in p.Leu775Valfs32, p.Thr1108Metfs8, and p.Pro5Argfs*2. : This study presents a comprehensive characterization of the targeted gene panel used for genetic analysis, while critically evaluating its diagnostic limitations within the context of contemporary genomic approaches. A pivotal accomplishment of this study was the ClinVar submission of novel de novo variants which expands the documented mutational spectrum of ASD-associated genes and enhances future diagnostic interpretation.