The sensitivity of the compendial European Pharmacopoeia RPLC/UV method for assaying residual fatty acids in Ginkgo biloba standardized extracts was essentially improved by operating modifications on the sample preparation procedure, including sample injection. The acidified methanol/water solution of the Ginkgo biloba standardized extract was extracted "in situ" with n-hexane (concentration factor of 20). From the organic layer, a relatively large volume (50 μL) of the hexane solution was directly loaded to the chromatog. column. A modified gradient elution profile was used to force the sample solvent to elute faster than analytes, without affecting peak symmetry. The separation is obtained in 22 min. As the amount of the target compounds loaded to column is higher, selective UV detection at 310 nm was possible. The method was validated according to specific operating guidelines for selectivity, linearity range, quantitation limits, precision, accuracy, and robustness. A limit of quantitation of 1 ppm (five times less than the accepted maximal content threshold for fatty acids in standardized G. biloba extracts) was obtained. Confirmation of the target compounds through MS2 detection and evaluation of the sensitivity afforded by such a detection system are also discussed.