In this study, we developed a streamlined method to efficiently generate conditional alleles in zebrafish using the invertible gene trap cassette Zwitch. We demonstrated that Zwitch can be inserted into a defined locus of the zebrafish genome via precise in vivo genome editing for inducible Cre-mediated gene disruption. In theory, the approach established in this study can be used to generate conditional alleles of any zebrafish gene, and it will expand the utility of zebrafish as a model organism.