HIV/AIDS remains a major health threat despite significant advances in the prevention and treatment of HIV infection. The major reason is the inability of existing treatments to eradicate the multiple HIV reservoirs in the human body, including astrocytes in the human brain. CRISPR/Cas9 system is an emerging gene-editing technique with the potential to eliminate or disrupt HIV provirus in HIV reservoir cells, which may lead to a complete cure of HIV/AIDS. The key components of CRISPR/Cas9 are guide RNAs (gRNAs) which determine specific sequence targeting of DNAs. This study established a novel, simple and quick screening method to identify gRNA candidates for targeting HIV provirus in astrocytes. Briefly, stable astrocytes clones with an integrated fluorescent HIV reporter and Cas9 expression gene were generated. Various gRNAs were screened for their efficiencies against HIV provirus in these cells. Moreover, these gRNAs and Cas9 protein were successfully tested on HIV latent astrocytes without Cas9 expression to mimic clinical conditions. HIV provirus gene-editing were confirmed by cell genomic DNA PCR and fluorescent marker expression analysis. In the future, the established transgenic cells can be used for other gene-editing studies and is well-suited for high-throughput screen application.