| Summary: | The major groups of antioxidant compounds (isoflavonoids, xanthones, hydroxycinnamic acids) in the rhizome methanol extracts of four Ukrainian <i>Iris</i> sp. (<i>Iris pallida</i>, <i>Iris hungarica</i>, <i>Iris sibirica</i>, and <i>Iris variegata</i>) were qualitatively and quantitatively analyzed using HPLC-DAD and UPLC-MS/MS. Gallic acid, caffeic acid, mangiferin, tectoridin, irigenin, iristectorigenin B, irisolidone, 5,6-dihydroxy-7,8,3′,5′-tetramethoxyisoflavone, irisolidone-7-<i>O</i>-<i>β</i>-d-glucopyranoside, germanaism B, and nigricin were recognized by comparing their UV/MS spectra, chromatographic retention time (tR) with those of standard reference compounds. <i>I. hungarica</i> and <i>I. variegata</i> showed the highest total amount of phenolic compounds. Germanaism B was the most abundant component in the rhizomes of <i>I. variegata</i> (7.089 ± 0.032 mg/g) and <i>I. hungarica</i> (6.285 ± 0.030 mg/g). The compound analyses showed good calibration curve linearity (r<sup>2</sup> > 0.999) and low detection and quantifications limit. These results validated the method for its use in the simultaneous quantitative evaluation of phenolic compounds in the studied <i>Iris</i> sp. <i>I. hungarica</i> and <i>I. variegata</i> rhizomes exhibited antioxidant activity, as demonstrated by the HPLC-ABTS system and NRF2 expression assay and anti-inflammatory activity on respiratory burst in human neutrophils. Moreover, the extracts showed anti-allergic and cytotoxic effects against cancer cells. Anti-coronavirus 229E and lipid formation activities were also evaluated. In summary, potent antioxidant marker compounds were identified in the examined <i>Iris</i> sp.
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