Advances in sequencing technologies have enabled the rapid identification of human disease genes by GWAS or whole exome sequencing techniques. There is a large-gap between identification of human disease genes and their functional validation. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9 including a variety of tools and methods for guide RNA synthesis and mutant identification. While all the published techniques work, not all approaches are readily scalable to increase throughput. In addition, zebrafish have been shown to effectively recapitulate disease phenotypes, however models that fail to generate relevant phenotypes are rarely reported. We recently described a CRISPR/Cas9 based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here we present a complete workflow including target selection, cloning-free single guide RNA (sgRNA) synthesis, microinjection, validation of the target-specific activity of the sgRNAs, founder screening to identify germline transmitting mutations, determination of the exact lesion by PCR and next generation sequencing (including software for analysis), and genotyping in the F1 or subsequent generations. We used this high throughput pipeline to target all published human genes linked to non-syndromic deafness in zebrafish. We will present phenotyping data from homozygous mutants, and determine a rate of successful modeling in zebrafish.