The side population (SP) cells are identified through Hoechst 33342 staining and analyzed using flow cytometry (FCM). The Hoechst SP method is utilized for the isolation of stem cells based on the dye efflux properties of ATP-binding cassette (ABC) transporters. This method was initially employed for the identification and isolation of hematopoietic stem cells (HSCs), but it has now evolved to primarily focus on the identification and isolation of cancer stem cells (CSCs). In this study, the capability of the 375 nm and 405 nm lasers in detecting SP cells was assessed and compared with that of the 355nm laser. Our findings demonstrate a remarkable similarity between the SP cells detected by the 355nm laser and the 405nm laser on the CytoFLEX system of FCM. These results suggest that the high-power 375 nm and 405 nm lasers can serve as a viable alternative to the 355 nm laser for SP cell detection.