The present study was undertaken to evaluate the in vivo analgesic activities of the extracts prepared from the aerial parts and roots of Scrophularia kotscyhana and to isolate the bioactive metabolites from the most active extract. Analgesic activities of all extracts and subextracts at the doses of 5, 10 and 30 mg/kg (i.p.) were examined using hot plate test in mice. Among the tested extracts, MeOH extract prepared from the aerial parts and the n-butanol subextract prepared thereof displayed the best analgesic activity at all doses. Phytochemical studies on n-butanol subextract led to the isolation of two new iri doi d glycosides as an inseparable mixture, 8-O-acetyl-4'-O-(E)-(p-coumaroyl)-harpagide (1) and 8-O-acetyl-4'O-(Z)-(p-coumaroyl)-harpagide (2) along with five known secondary metabolites, beta-sitosterol 3-O-beta-glucopyranoside (3), apigenin 7-O-beta-glucopyranoside (4), apigenin 7-O-beta-rutinoside (5), luteolin 7-O-beta-glucopyranoside (6) and luteolin 7-O-rutinoside (7). The iridoid mixture (1 and 2), 3 and 4 elicited significant inhibition of pain at 5 mg/kg dose.