The ethanolic extract of Nectandra reticulata contains a high amount of quercetin-3-O-rhamnoside (quercitrin) that has exhibited a significant activity toward Alzheimer's disease, specifically with LXR receptors. In this work, a methodology was validated following the specifications of the International Conference of Harmonization in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), repeatability (intra-assay), intermediate precision (intra-laboratory), reproducibility (inter-laboratory), robustness, and specificity. The effect of location (Oiba, Granada, and Chiquinquira) and the extraction method (percolation, maceration, and ultrasound-assisted extraction) towards the chromatographic profile and quercitrin recovery was studied. Furthermore, a Box-Behnken design was conducted to optimize quercitrin extraction and extraction yield by ultrasonic-assisted extraction. The chromatographic method was validated, with a linear range from 5 to 180 mg quercitrin per L, LOD 0.26 mg L-1, and LOQ 0.86 mg L-1. Accuracy [recovery of 93.8% (w/w)], repeatability (relative standard deviation, RSD, 3.3%), intermediate precision (RSD 5.4%), and reproducibility (RSD 1.4%) were within the acceptable values. The method was robust and specific, except for the variation in the formic acid concentration. The location had a greater influence than the extraction method towards both the chromatographic profile and quercitrin recovery. Quercitrin extraction was maximized at 60% (v/v) ethanol and 50 °C, independent of the solvent : material ratio used. The highest yield values were achieved at 60% (v/v) ethanol and 50 °C, with a solvent : material ratio of 40 mL g-1.