Ovarian cancer (OC) accounts as a common critical malignant tumor among female diseases. Among numerous drug resources, botanical compounds identified and isolated from plants have unique advantages due to their potential as chemotherapeutic drugs for cancer treatment. The aim of the study is to apply innovative extraction technique to increase carpaine obtained yield and investigate its antiproliferative activity on ovarian cancer. This work reports the extraction of carpaine from the leaves of Carica papaya using microwave-assisted extraction (MAE). Quantitative analysis of carpaine in the ethanol extract was conducted using HPLC with UV detection. The antiproliferative activity of extracted carpaine was investigated on CHO-K1 cell line (ovarian carcinoma) by MTT assay. Molecular modeling was employed to check the binding mode of carpaine with the 20S proteasome enzyme. Carpaine was detected in the alkaloid residue at 304 nm with retention time 36.50 min and the HPLC analytical methodology for standard curve was developed and validated to quantify carpaine as 0.29% of the dried leaves of Carica papaya. It was highly active against the human ovarian cancer cell line, CHO-K1, with IC50 value of 28.40±3.2 μg/ml compared to vinblastine sulfate, the reference standard, which gave IC50 value of 9.65±0.74 μg/ml. Molecular study emphasized the influential carpaine fitting in the binding site of 20S proteasome enzyme suggesting it is the prospective key player for carpaine’s observed anti-ovarian cancer activity. These results suggest important guidance for the potential pathway of carpaine as an effective Carica papaya’s extract component for the prevention or amelioration of cancer. Consequently, it is highly expected to be utilized as functional food contributing to the preservation and promotion of human health. © 2023 National Information and Documentation Center (NIDOC)