ABSTRACT: Fruits and vegetables are considered a good source of antioxidants, which are beneficial in protecting the human body against damage induced by free radicals and other reactive oxygen (ROS) and nitrogen (RNS) species. In this work, we aimed to evaluate the integral antioxidant activity (AOA) and determine individual polyphenols in fruits and vegetables of frequent consumption. For this purpose, an innovative and high throughput analytical approach based on original QuEChERS assisted by ultrasound extraction (USAE), instead of the manual agitation used in the classical procedure, was optimized and implemented for the isolation of polyphenols. The total phenolic content (TPC), flavonoids, anthocyanins, and betalains were evaluated using different spectrophotometric assays. In addition, free radical scavenging by methods 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) were used to estimate the AOA of the investigated fruit and vegetable extracts. Red onion, tamarillo, and beetroot were the samples with the highest AOA. The quantification and identification of free low molecular weight polyphenols from QuEChERS-USAE extracts was carried out by ultra-high-pressure liquid chromatography equipped with a photodiode array detection system (UHPLC-PDA). Catechin was the most abundant polyphenol, followed by gentisic and ferulic acids, mainly in the watercress sample. In relation to flavonols, quercetin and kaempferol were found mostly in onion samples, and in small quantities in tomato and watercress. The improved analytical approach, QuEChERS-USAE/UHPLC-PDA, offers an attractive alternative for the analysis of polyphenols from fruit and vegetable samples, providing several advantages over traditional extraction techniques, in terms of reproducibility, simplicity, low cost, analysis speed, and analytical performance.