AbstractFlavonoids are a class of specialized metabolites found in many different plant species. They protect against UV radiation, scavenge reactive oxygen species, are involved in plant defense responses, and are associated with plant‐microorganism interactions. They have also been reported to possess health‐promoting effects including anti‐inflammatory, antioxidant, anticancer activity, and antihypertensive effects. Flavonoids encompass >10,000 structures where the types and amounts depend on the plant species, developmental stage, organ, and growth conditions. The diversity of flavonoid structures represents a significant challenge in the analysis of plant flavonoids. Many analytical techniques have been developed to detect and quantify flavonoids, and the most productive of these techniques use liquid chromatography (LC) coupled to mass spectrometry (MS) to analyze flavonoids due to the excellent combination of selectivity and sensitivity of MS. In addition, mass spectral libraries have been constructed to further aid flavonoid identification. Here, the use of ultra‐high pressure liquid chromatography coupled to mass spectrometry (UHPLC‐MS) in plant flavonoid analyses, with an emphasis on sample extraction, flavonoid separation, and MS detection, is described. © 2018 by John Wiley & Sons, Inc.