High-performance liquid chromatography (HPLC) is one of the most used liquid chromatographic techniques owing to its versatility that allows an optimal resolution. Quercetin (QUE), a polyphenol, has received increasing attention due to its antioxidant properties. Efforts have been made to overcome the traditional poor oral bioavailability of quercetin, requiring the development of analytical techniques for quercetin detection in different matrices. The main objective of this work was the optimization of a HPLC-Diode Array Detector (DAD) method for quercetin. An optimization process was conducted to guarantee the best conditions and results. Detection and quantification were executed with a reverse phase C-18 column. Different mobile phase (MP) compositions, flow rates, and wavelengths (with DAD) were studied. Better results were achieved with a MP composition of water:acetonitrile:methanol (55:40:5), with 1.5% acetic acid at a 1.3 mL/minute flow rate, with good results regarding chromatogram resolution, and quercetin quantification and detection.