Objective: To evaluate the cytotoxic activity of wood extracts of Lunasia amara Blanco (L. amara) and to perform further phytochemical standardization. Methods: The wood extracts of L. amara were assessed for cytotoxic activity by in vitro tetrazolium bromide (MTT) method against two human cancer cell lines, cervical cancer cells (HeLa) and breast cancer cells (T47D). Thin layer chromatography, Dragendorf, acetic anhydride-sulfuric acid and ferric chloride were used to detect alkaloids, steroids and polyphenols, respectively. Furthermore, quantitative determination of total alkaloid by ultra fast liquid chromatography-photodiode array detection using lunacrine as a marker compound was performed as well. Results: The ethyl acetate extract exhibited higher cell-growth inhibition than methanol and n-hexane extracts on HeLa and T47D cancer line cells with the IC₅₀ of 71.15 and 79.04 μg/mL, respectively. Total alkaloid in ethyl acetate extract was counted as (10.46 ± 0.28)% (w/w), while lunacrine determined by ultra fast liquid chromatographyphotodiode array detection method was found to be (3.55 ± 0.26)% (w/w). Conclusions: The high total alkaloid and lunacrine concentration on the extract confirm the potential cytotoxic property of ethyl acetate wood extract of L. amara.