A simple, rapid, and sensitive LC method to determine coumestrol incorporated in the lipid nanoemulsions was validated. The analyses were performed at room temperature on a reversed-phase C18 column using a mobile phase composed of methanol/water with 0.1% trifluoracetic acid (70:30, v/v) at 0.8 mL min(-1). The detection was carried out on a UV detector at 343 nm. The linearity, in the range of 0.1-6.0 microg/mL, presented a determination coefficient (r2) of 0.999, calculated by the least square method. No interferences of the oil core or the gelling excipients were detected. The R.S.D. values for intra- and inter-day precision experiments were lower than 2%. The recovery ranged from 99.42% to 100.72%. Finally, the proposed method was successfully applied to determine coumestrol incorporated in the proposed topical formulations.