This study describes the production of calli from Globularia trichosantha subsp. trichosantha and the quantitative determination of catalpol, aucubin, and verbascoside in the calli and the plant by HPLC. The seeds of the plant were sterilized and germinated in Murashige and Skoog (MS) medium without in vitro plant growth regulator. Hypocotyl, cotyledon, first leaf, epicotyl, apical meristem, and root explants were taken from the 30-day-old aseptic seedlings germinated in vitro. Explants were then transferred to MS media for callus production together with varying concentrations of plant growth regulators. The best callus production occurred in the media containing 6 mg L-1 IAA and 0.2 mg L-1 2,4-D + 0.1 mg L-1 BAP. The highest catalpol, aucubin, and verbascoside contents were determined in the roots (1.277 mg kg(-1)), stem (0.775 mg kg(-1)), and roots (0.290 mg kg(-1)), respectively. The study is important in the sense that, 1) it is the first tissue culture study on G. trichosantha subsp. trichosantha that provides basic information for callus production; 2) that catalpol, aucubin, and verbascoside were quantitatively determined in calli and plant for the first time; and 3) all the secondary metabolites studied here were produced in the callus culture in a quantity that is approximately 7 times higher than the amount that naturally occurs in the plant.