Abstract: Objective：To investigate the impact of different concentrations of Hoechst 33342 and verapamil on the side population (SP) cells sorted by flow cytometry. Methods：The human lung adenocarcinoma A549 cells were treated by different concentrations of Hoechst 33342 (2.5, 5.0 and 7.5 μg/mL) and the Hoechst 33342 combined with 50, 100 or 150 μmol/L of verapamil, respectively, and sorted the SP cells by flow cytometry in order to obtain the optimal concentration of Hoechst 33342 and verapamil. Then, the tumor cell line K1 was used to verify the optimal concentration of Hoechst 33342 and verapamil. Results：When the concentrations of verapamil and Hoechst 33342 were 150 μmol/L and 7.5 μg/mL, respectively, the A549 cells were stained completely, and SP cells and non-SP cells were classified obviously. The SP cells accounted for about 2.2%. When the concentrations of verapamil and Hoechst 33342 were 50 μmol/L and 5.0 μg/mL, respectively, the SP cells and non-SP cells of K1 cells were classified obviously, and the SP cells accounted for about 1.3%. The sorted SP cells and non-SP cells could be further cultured and passaged. Conclusion：The concentration of Hoechst 33342 and verapamil may influence the sorting efficiency of SP cells in tumor cells.