This study was carried out to optimize the various approaches to analyze the effects of various variables on the total flavonoid content extraction from the roots of Helicteres hirsuta L. The existence of various compounds in the methanol fraction was accessed by using LC-MS/MS analysis. The results of the study identified the ideal parameters such as times (30 minutes); methanol solvent concentration (50%); ultrasonic frequency (12 Hz); and material/solvent ratio [1:30 (w/v)] for extracting the highest total flavonoids from the roots of H. Hirsuta. The study's results suggested that the total flavonoid value was 3.52684 (mg Catechin/g extract). The verified experiment obtained an actual value of 5.205 (mg Catechin/g extract). Further, the results of the study suggested the presence of 20 compounds of a flavonoid nature (66.667%) appearing in the purified methanol fractional extract. These compounds can inhibit DPPH free radicals at 50%, with an IC50 value of 536.760 g/mL, and they also have inhibitory activity on the growth of cancer cell lines with IC50 values ranging from 115.81 and 219.17g/mL. The human leukemia cell line (HL-60) exhibits the most significant cytotoxic response to a methanol extract from H. hirsuta root with an IC50 value of 115.81 g/mL.