Summary A reliable and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detection method was developed and validated for the quantification of hopantenic acid in human plasma. Hopantenic acid, with protocatechuic acid as the internal standard (IS), was extracted from plasma samples using a liquid-liquid extraction with methanol. A chromatographic separation was achieved on a Luna C18 column (4.6 mm × 150 mm, 5-μm particle size) and precolumn of the same sorbent (2.0 mm). An isocratic elution, at a flow rate of 1.0 mL min−1, was used with a mobile phase consisting of acetonitrile, water, and 0.03% trifluoroacetic acid. The UV detector was set to 205 nm. The elution times for hopantenic acid and IS were ∼4.3 and 5.4 min, respectively. The calibration curve of hopantenic acid was linear (r > 0.9994) over the range of 0.5–100 μg mL−1 in human plasma. The limit of detection and limit of quantification for hopantenic acid were 0.034 and 0.103 μg mL−1, respectively....