The purpose of this study was to analyze the HPLC-DAD-ESI-Q-TOF-MS/MS profile of the methanolic extract obtained from the aerial parts of Verbascum blattaria L. and develop efficient high-performance countercurrent chromatography (HPCCC)methods for isolation of its major iridoid glycosides. Additionally, the potential anti-inflammatory activities of the extract and two of the isolated compounds were evaluated using a human neutrophil model. LC–MS analysis led to the identification of 35 constituents, of which 18 iridoids, eight saponins, three phenylethanoids, four flavonoids and two phenolic acids. Scropolioside F (A, 2 mg), scrophuloside A₃ (B, 16 mg)and gmelinoside L (C, 13 mg)were obtained from 300 mg of the crude methanolic extract by HPCCC with the help of two different biphasic solvent systems: n-hexane/ethyl acetate/n-butanol/water (1/2/1/2; v/v/v/v)and n-hexane/ethyl acetate/methanol/water (1/19/1/19; v/v/v/v). The crude methanolic extract (100 μg/mL)down-regulated lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α)release in a comparable extent to dexamethasone (25 μM). Gmelinoside L (50 μM)was more active than scrophuloside A₃, producing inhibition percentages of 43.52% and 41.28% on interleukin 8 (IL-8)and TNF-α level, respectively. The HPCCC methods developed in the current work may serve to large scale isolation of iridoid glycosides from Verbascum species or other iridoid abundant-genera.