Gene expression profiling is a powerful technique for studying biological processes, especially tissue/organ-specific ones, at the molecular level. With the rapid development of the next-generation sequencing techniques, high throughput sequencing-based expression profiling techniques have been more and more widely adopted in molecular biology studies. In this chapter, we described a protocol for applying one of the sequencing-based expression profiling techniques, Digital Gene Expression (DGE), for zebrafish research. The protocol provides guidelines for wet-bench experimental procedures as well as for bioinformatics data analyses. We also discuss potential issues/challenges with the use of DGE.