Rauvolfia tetraphylla is an essential medicinal plant that has been widely used in traditional medicine for various disease treatments. However, the tumor suppressor activity of R. tetraphylla and its phytocompounds were not explored against triple-negative breast cancer. The current research investigated the impact of R. tetraphylla methanolic extract (RTE) and its isolated compounds Ajmaline (RTC1) and Reserpine (RTC2) on triple-negative breast cancer cell line (MDA-MB-231) focusing on anti-proliferative effects. Our study imparts that RTE and RTC2 showed promising cytotoxic effects compared to RTC1. So further experiments have proceeded with RTE and RTC2, to evaluate its proliferation, migration, and apoptotic effect. The result shows around 80% of cells were observed in the G0/G1 phase in cell cycle analysis indicating the cell cycle inhibition and duel staining clearly showed the apoptotic effect. The migration of cells after the scratch was 60.45% observed in control and 90% in treated cells showing the inhibition of migration. ROS distribution was intense compared to control indicating the increased ROS stress in treated cells. Both RTE and RTC2-treated cells showed the potential to suppress proliferation and induce apoptotic change by upregulating BAX and MST-1 and suppressing Bcl2, LATS-1, and YAP, proving that deregulation of YAP resulting in the blockage of TEAD-YAP complex and inhibit proliferation. Therefore, R. tetraphylla extract and its isolated compounds were demonstrated to find its ability to act against MDA-MB-231 and these findings will help adjudicate it as a therapeutic drug against experimental triple-negative breast cancer.