Syndromic hearing loss accounts for approximately 30% of all cases of hearing loss due to genetic causes. Mutation screening in known genes is important because it potentially sheds light on the genetic etiology of hearing loss and helps in genetic counseling of families. In this study, we describe a customized Ion AmpliSeq Panel, specifically designed for the investigation of syndromic hearing loss. The Ion AmpliSeq Panel was customized to cover the coding sequences of 52 genes. Twenty-four patients were recruited: 17 patients with a clinical diagnosis of a known syndrome, and seven whose clinical signs did not allow identification of a syndrome. Of 24 patients sequenced, potentially causative mutations were found in nine, all of which belonged to the group with a previous clinical diagnostic and none in the group not clinically diagnosed. We were able to provide conclusive molecular diagnosis to six patients, constituting a diagnostic rate of 25% (6/24). In the group of patients with a suspected clinical diagnosis, the diagnostic rate was 35% (6/17). Of the nine different mutations identified, three are novel, and were found in patients with Waardenburg, Treacher Collins and CHARGE syndromes. Since all patients with a conclusive molecular diagnosis through this panel had a previous suspected clinical diagnosis, our results suggest that this panel was more effective in diagnosing this group of patients. Therefore, the panel demonstrated effectiveness in molecular diagnosis when compared to others in the literature, especially for patients with a defined clinical diagnosis.