This article reports the validation of a spectrophotometric method to estimate coumarins incorporated into nanoemulsions containing Pterocaulon balansae Chodat extract. The quantification was based on the assay of esculin at 327 nm, which presents the same substitution pattern of coumarins isolated from Pterocaulon species. Linear response (R2 > 0.995) was observed over the range of 5.0 to 25.0 μg/mL. The relative standard deviation values for the intra- and inter-days precision were lower than 3.0 %. The recovery ranged from 93.3 % to 104.1 %. The association efficiency was estimated after the determination of free coumarins in the water phase of nanoemulsions obtained after separation through ultrafiltration/centrifugation devices. The coumarins association was approximately 92 %.