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.