The unparalleled heterogeneity in genetic causes of hearing loss along with remarkable differences in prevalence of causative variants among ethnic groups makes single gene tests technically inefficient. Although hundreds of genes have been reported to be associated with nonsyndromic hearing loss (NSHL), GJB2, GJB6, SLC26A4, and mitochondrial (mt) MT-RNR1 and MTTS are the major contributors. In order to provide a faster, more comprehensive and cost effective assay, we constructed a DNA fluidic array, CapitalBioMiamiOtoArray, for the detection of sequence variants in five genes that are common in most populations of European descent. They consist of c.35delG, p.W44C, p.L90P, c.167delT (GJB2); 309kb deletion (GJB6); p.L236P, p.T416P (SLC26A4); and m.1555A>G, m.7444G>A (mtDNA). We have validated our hearing loss array by analyzing a total of 160 DNAs samples. Our results show 100% concordance between the fluidic array biochip-based approach and the established Sanger sequencing method, thus proving its robustness and reliability at a relatively low cost.