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Phyllanthus niruri L. is one of the wild species originated-medicinal plant. It is widely used for various purposes, such as immunostimulant, anti-hepatotoxic, anti-HIV, anti-lithic, anti-hypertensive, and anti-hepatitis B. One of its significant phytochemicals contents is a flavonoid. Cultivation method, harvesting time, and flavonoid extraction time were estimated to have different responses on P. niruri L. flavonoid content. Therefore, this research was carried out to investigate the effect of cultivation, harvesting time, and extraction time on total flavonoid content by colorimetric method using AlCl3 reagent and to obtain the optimum harvesting time and extraction time for the highest total flavonoid content of P. niruri L. The experiment was conducted at South Tangerang, Banten, Indonesia, and designed in 3 factors of completely randomized block design with four replications. The first factor was cultivation type (seeding on an organic tray, seeding on a plastic tray, direct seeding on the field with mulch, and direct seeding on the field without mulch). The second factor was harvesting time (7, 8, and 9 Weeks After Planting-WAP), and the third factor was P. niruri L. methanol extracted time (1 hour, one week, two weeks, three weeks, and four weeks). The results showed that the cultivation, harvesting, and extraction time significantly affected the total flavonoid content. No significant interaction was revealed in this experiment. The highest flavonoid content was shown by direct seeding on the field with mulch, harvesting time at 8 WAP, and two weeks flavonoid extraction period. [ABSTRACT FROM AUTHOR]