Tagetes erecta flowers contain nematoside compound α-Terthienyl (2,2′:5′,2″-Terthiophene) which are toxic to erythrocytes and human skin. In this study, analysis of the content of α-Terthienyl (2,2′:5′,2″-Terthiophene) in orange and yellow Tagetes erecta flowers originating from several areas in West Java by HPLC and LC-MS/MS have been studied. The flowers are dried in an oven at 45 °C, then crushed in a blender and sieved with a 0.35 μm sieve. The flower powder is then extracted with n-hexane using a waterbath shaker at 65 °C for 1 h. The solvent extraction then evaporated in a water bath until dry. The residue in the form of a thick extract is dissolved with ethanol, then filtered with a 0.45 μm membrane filter, then injected into the HPLC system. This filtered solution was also diluted 20 times with acetonitrile for analysis by LC-MS/MS. Content of α-Terthienyl (2,2′:5′,2″-Terthiophene) obtained from the HPLC and LC-MS/MS analysis were not significantly different. The α-Terthienyl (2,2′:5′,2″-Terthiophene) peak is well separated from the other peaks. Orange flowers from each region contain higher α-Terthienyl (2,2′:5′,2″-Terthiophene) than yellow flowers. This method is sensitive and easy to do because it does not require further purification of the extract and uses a simple mobile phase.