Derris elliptica which is known locally as 'tuba' plant contains rotenone, a well-known bio-active compound that has the potential to be used as bio-pesticide. Bio-pesticide is the best-known alternative to the commercially available conventional pesticides and it is more environmental friendly. For that reason, the aim of this study is to obtain the best extraction solvent system that produces high yield of rotenone and to determine the rotenone content with respect to different locations of Derris roots in Johor, Malaysia. Rotenone was extracted using normal soaking extraction (NSE) process using three different parts of Derris plant and three types of solvent system. The types of Derris part were: (1) fine root, (2) coarse root and (3) stem. The types of solvent system were: (1) acetone 95% (v/v), (2) Chloroform 99.9% (v/v) and (3) a mixture of ethanol and water (9:1) added with oxalic acid (1 mg/ml). Next, the liquid crude extracts were filtered to remove any fine debris prior to the reverse-phase high performance liquid chromatography (RP-HPLC) analysis. The results showed that acetone 95% (v/v) was the best solvent system to extract the highest yield of rotenone (1.14% w/w) of Derris fine roots. Meanwhile, Kangkar Pulai was the best location to procure this domesticated forest Derris plants with high content of rotenone due to the fact that the location where it thrived was nearby to the river stream, shady spot and humid condition. For that reason, the yield of rotenone was highly affected by location, types of solvent and plant parts used. Keywords: Derris elliptica, Rotenone, Bio-pesticide, Normal Soaking Extraction (NSE)