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A reproducible analytical method using reverse-phase high liquid performance chromatography combined with UV detecting was developed for the quantitative determination of four compounds isolated from the ethanol extract of Phaseolus angularis seeds (PASE): oleanolic acid (1), oleanolic acid acetate (2), stigmasterol (3) and β-sitosterol (4). This method was fully validated in terms of linearity (r2 > 0.999), accuracy (98.5%–100.8%), precision (<0.92%), LOD (<0.0035 mg/mL), and LOQ (<0.0115 mg/mL). The effects of the PASE and isolated compounds 1–4 on TLR4 activation were tested in THP1-Blue cells. Among the tested substances, compound 2 showed potent inhibitory activity with an IC50 value of 3.89 ± 0.17 µM.