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A reversed-phase high-performance liquid of chromatography (RP-HPLC) method for determination of two isomer sesquiterpene lactone deoxyelephantopin (1) and isodeoxyelephantopin (2) from Elephantopus scaber L. was developed and validated. Separation was achieved on a Phenomenex Luna C-18 (250 × 4.6 mm, 5 μm) column, with water: acetonitrile: 2-propanol (66:20:14, v/v/v) as mobile phase, flow rate of 1.0 mL-1 and UV detection was set up at 210 nm using DAD detector. Linearity for both compounds was found in the concentration range of 0.251-1.506 and 0.516-3.096 μg mL-1 (r2 ≥ 0.99), LOD is 0.094 and 0.151 μg mL-1, LOQ is 0.285 and 0.457 μg mL-1, and the range of recovery is 97.64-104.98% and 95.23-102.25%, respectively. The results of intra- and inter-day validation (n=3) showed the method was efficient and could be applied for the determination of sesquiterpene lactones in Elephantopus scaber L. extract.