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[Display omitted] • Optimum UAE conditions occurred at amplitude (10 %–70 %) and treatment time (2–10 min) on both responses, i.e., TPC and TFC of P. indica (L.) leaves. • The optimum P. indica (L.) leaf extract had positive cytotoxicity on the HT-29 CRC cell line, in which the IC50 was 12 µg/mL. • The optimum P. indica (L.) leaf extract contained flavonols, namely, kaempferol 3-[2′'',3′'',5′''-triacetylalpha-L-arabinofuranosyl-(1->6)-glucoside, myricetin 3-glucoside-7-galactoside, quercetin 3-(3′'-sulfatoglucoside), and kaempferol 7,4′-dimethyl ether 3-O-sulfate, which were the major components. Colorectal cancer (CRC) is the most common malignancy and the third primary cause of cancer-related mortalities caused by unhealthy diet, hectic lifestyle, and genetic damage. People aged ≥ 50 are more at risk for CRC. Nowadays, bioactive compounds from plants have been widely studied in preventing CRC because of their anticancer and antioxidant properties. Herein, ultrasound-assisted extraction (UAE) was used to extract the bioactive compounds of Pluchea indica (L.) leaves. The resultant total phenolic content (TPC) and total flavonoid content (TFC) of P. indica (L.) leaves were analyzed using a response surface methodology (RSM). The central composite design was implemented to evaluate the amplitude (10 %–70 %) and treatment time (2–10 min) on both responses, i.e., TPC and TFC of P. indica (L.) leaves. The optimum UAE conditions were observed 40 % amplitude and 6 min of treatment, where the TPC and TFC were 3.26 ± 0.00 mg GAE/g d.w. and 67.58 ± 1.46 mg QE/g d.w., respectively. The optimum P. indica (L.) leaf extract was then screened for its cytotoxicity on the HT-29 colorectal cancer cell line. This extract had strong cytotoxicity with a half-maximal inhibitory concentration value (IC 50) of 12 µg/mL. The phytochemical screening of bioactive compounds revealed that the optimal P. indica (L.) leaf extract contains flavonoids, namely, kaempferol 3-[2′'',3′'',5′''-triacetyl]-alpha-L-arabinofuranosyl-(1->6)-glucoside, myricetin 3-glucoside-7-galactoside, quercetin 3-(3′'-sulfatoglucoside), and kaempferol 7,4′-dimethyl ether 3-O-sulfate, which could be good sources for promising anticancer agents. This study employs the RSM approach to utilize UAE for bioactive compounds extraction of P. indica (L.) leaves, identified the specific compounds present in the optimized extract and revealed its potential in preventing CRC. [ABSTRACT FROM AUTHOR]