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A simple, rapid, specific, and sensitive method was developed for the simultaneous identification and quantification of six major bioactive compounds, namely, caffeic acid, quercetin, apigenin, ferulic acid, baicalein, and kaempferol, from Asparagus officinalis roots (ARs) native to New Zealand (green and purple cultivars) and China (yellow, green, purple, and white cultivars) using ultrasound-assisted, solid-phase extraction (UASE-SPE) coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The method was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy (expressed as recovery %), and precision (expressed as relative standard deviation (%RSD)). The retention times, ultraviolet visible (UV-vis) data, and mass spectral patterns of the detected peaks matched those of commercial standards, allowing characterization of the target compounds. The LODs and LOQs were 23 ng/mL and 70 ng/mL, 50 ng/mL and 150 ng/mL, 10 ng/mL and 30 ng/mL, 18 ng/mL and 54 ng/mL, 14.4 ng/mL and 43.6 ng/mL, and 7.5 ng/mL and 22.5 ng/mL for caffeic acid, quercetin, apigenin, ferulic acid, baicalein, and kaempferol, respectively, and the mean recovery rates were 85.8%, 73.0%, 90.2%, 80.6%, 76.7%, and 74.5% for the six compounds, respectively. The levels of the target compounds were significantly different ( p < 0.05) among the six cultivars. The Chinese yellow AR had the highest levels of bioactive compounds: 6.0, 3.9, 0.4, 1.0, 0.86, and 0.8 mg/g for caffeic acid, quercetin, apigenin, ferulic acid, baicalein, and kaempferol, respectively. The AR extracts showed protective effects against oxidative stress in the HepG2 and L929 cell lines. The results indicate that AR extracts contain high flavonoid levels that provide protective functions against oxidative stress and support the potential commercial application of AR extracts.