The　effects　of　ultrasonic　parameters　and　treatment　conditions　on　the　in　vitro　cellular　experiments　of　sonodynamic　therapy　(SDT)　have　not　been　fully　studied.　Exploring　the　factors　that　affect　the　efficacy　of　SDT　can　provide　a　reference　for　screening　effective　sonosensitizers　in　vitro.　The　aim　of　this　work　is　to　investigate　the　factors　that　affected　the　SDT　effects　in　cancer　cells.　Cancer　cells　in　culture　plates　were　exposed　to　ultrasound　and　sonosensitizers.　The　intracellular　drug　concentration　was　measured　by　using　flow　cytometry　and　the　cell　viability　was　determined　by　MTT　assay.　The　SDT　effects　of　cancer　cells　treated　with　different　ultrasonic　parameters　under　the　same　sonosensitizer　concentration　were　different.　The　ultrasonic　parameters,　intracellular　drug　concentration,　drug　treatment　time,　cell　amount,　and　cell　status　could　affect　the　sonodynamic　therapeutic　effects.　It　is　necessary　to　select　appropriate　ultrasound　conditions　and　optimize　the　cellular　status　to　make　the　results　of　the　in　vitro　cellular　experiments　more　reliable.　©　2023,　The　Author(s),　under　exclusive　licence　to　Springer　Science+Business　Media,　LLC,　part　of　Springer　Nature.