A valuable and widely utilized approach to study gene regulatory networks is through transgenic lines. However, detailed information of transgene insertion sites is often not available, complicating the interpretation of experimental results involving transgenes. Here, we present a protocol for mapping the insertion sites of zebrafish Tol2 transgenes, using the recently developed tagmentation-based TransTag technique. We describe detailed steps for preparing sequencing libraries and analyzing raw reads. This protocol can be used to robustly identify transgene insertion sites in zebrafish. For complete details on the use and execution of this protocol, please refer to Meng et al.¹.